Ethoxyresorufin O-deethylase Activity Research Articles

Biomonitoring of polycyclic aromatic hydrocarbons (PAHs) from Manila clam Ruditapes philippinarum in Laizhou, Rushan and Jiaozhou, bays of China, and investigation of its relationship with human carcinogenic risk

This study examined the marine environment and seafood safety using chemical monitoring and multiple biomarkers. Samples were collected from three bays on the Shandong Peninsula in China, Laizhou, Rushan and Jiaozhou, in March, May, August, and October of 2018 and 2019. The polycyclic aromatic hydrocarbon (PAH) concentrations in sediments and tissue samples from the clam Ruditapes philippinarum and multiple biomarkers were measured. All the sampling sites were found to be medium-PAH-contaminated areas (100–1000 ng/g d.w.). According to the correlation analysis, ethoxyresorufin-o-deethylase (EROD) and superoxide dismutase (SOD) activity in the clam's digestive gland were sensitive to PAHs (p < .05), but the incremental lifetime cancer risk (ILCR) was lower than the priority risk level (10−4) at most sampling sites. EROD, SOD and acetylcholinesterase activity exhibited significant correlations with the ILCR values (p < .01), suggesting that they may serve as good indicators for assessing safe seafood consumption levels for human beings.

Monitoring of pollution in the western Black Sea coast of Turkey by striped red mullet (Mullus surmuletus).

The striped red mullet (Mullus surmuletus) is an economically important demersal fish species. In this study, our aim was to monitor the pollution in the western Black Sea coast of Turkey using striped red mullet as a bioindicator species. Fish samples were caught from four different locations in the western Black Sea coast of Turkey in 2006, 2009-2011, and 2016. Highly elevated cytochrome P4501A (CYP1A)-related 7-ethoxyresorufin O-deethylase (EROD) activities were measured in striped red mullet caught from Zonguldak Harbor in all of the sampling years. The lowest EROD activities were measured in fish samples caught from Kefken. In addition to the EROD activity measurements, glutathione S-transferase (GST), glutathione reductase, and catalase activities were also measured in the striped red mullet samples. Higher GST and catalase activities were measured in the striped red mullet samples caught from Zonguldak Harbor than from Kefken in 2016. These results indicate that the striped red mullet is responsive to CYP1A inducer pollutants. This study covers intermittent measurements of the biomonitoring data from the striped red mullet caught around the western Black Sea coast of Turkey, over a 10-year period.

Biological uptake, depuration and biochemical effects of diclofenac and carbamazepine in Carassius carassius

The uptake and depuration kinetics of diclofenac and carbamazepine alone at an environmentally relevant nominal concentration of 2 μg/L and in combination at a concentration ratio of 1:1 with total concentration of 4 μg/L were evaluated in Carassius carassius after 7 d uptake and depuration. Also, the biochemical effects of both drugs alone at nominal concentrations of 2 and 10 μg/L as well as in combination with total concentrations of 4 and 20 μg/L were investigated in Carassius carassius after 7 d exposure followed by 10 d recovery. In the single treatments, steady-state BCFs measured after the 7 d exposure were 73.05, 49.71, 38.01 and 24.93 L/kg for diclofenac and 9.25, 8.99, 5.29 and 4.11 L/kg for carbamazepine in the liver, brain, gill and muscle of Carassius carassius, respectively. Comparatively lower BCFs were measured in the tissues of Carassius carassius for both drugs in the combined treatments. Acetylcholinesterase activity in the brain was significantly induced by diclofenac while carbamazepine and the mixtures significantly inhibited it during all the exposure days as well as after the 10 d recovery in all treatments. This indicates that Carassius carassius could not recover from the neurotoxic effects caused by carbamazepine unlike the inductive effect caused by diclofenac which was recoverable after 10 days. A significant increase in the activities of 7-ethoxyresorufin O-deethylase and glutathione s-transferase for individual and mixed pharmaceuticals suggest that metabolism and detoxification of both drugs took place in the liver of Carassius carassius. Also, a significant increase in the activities of superoxide dismutase, catalase, glutathione reductase and malondialdehyde contents in the individual and mixture treatments mean that the antioxidant defence system of Carassius carassius was triggered to fight against oxidative stress but lipid peroxidation still occurred. However, Carassius carassius recovered from all these increases (superoxide dismutase, catalase, glutathione reductase and malondialdehyde) after the 10 d recovery, suggesting that oxidative damage is reversible. Our results indicate that both drugs at environmentally relevant concentrations might cause adverse effects in Carassius carassius and other fish species.

Biotransformation in the fish Prochilodus lineatus: An organ-specific approach to cyp1a gene expression and biochemical activity

The biotransformation ability of the organism is the result of organ-specific responses. This paper presents a molecular and biochemical approach to elucidate the biotransformation mechanisms in different organs of Prochilodus lineatus induced at 6, 24, and 96 h after a benzo[a]pyrene (B[a]P) injection. The induction in cyp1a transcription showed an organ-specific intensity at every tested time time. The EROD (ethoxyresorufin-O-deethylase) activity increased rapidly (6 h) in the liver and the kidney; the gills and the brain showed an increase at 24 h; and the gills demonstrated the highest activity among all the organs tested. There was no increase in glutathione S-transferase (GST) activity or lipoperoxidation. The decreased hepatic glutathione content (GSH) may be due to its role as an antioxidant. B[a]P was detected in the bile, confirming the xenobiotic efflux from the metabolizing organs. The gills, liver, brain, and kidney of P. lineatus presented an integrated mechanism to deal with the xenobiotic biotransformation.

The effect of co-administration of berberine, resveratrol, and glibenclamide on xenobiotic metabolizing enzyme activities in diabetic rat liver

It is possible to use plant-derived antioxidant molecules in the form of dietary supplements. However, dietary supplement–drug interaction pattern has not been well defined for most of these products. The aim of this study was to determine the effects of berberine, resveratrol, and glibenclamide on xenobiotic metabolizing enzyme activities in diabetic rats. Streptozotocin was administered to create experimental diabetes. Resveratrol (5 mg/kg) (R), glibenclamide (5 mg/kg) (G), and berberine (10 mg/kg) (B) were administered individually or in combinations in DMSO by intraperitoneal administration route to the diabetic rats. DMSO was also given to non-diabetic control (C) and diabetic control (D) groups. Livers of rats were taken under anesthesia at the end of the treatment period (12 days). Ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), aniline 4-hydroxylase (A4H), erythromycin N-demethylase (ERND), glutathione S-transferase (GST), catalase (CAT), and glutathione reductase (GR) activities were measured in microsomes and cytosols. In addition, histomorphological studies were also performed in the liver tissues. EROD activity of D+R was significantly higher than C and D+R+B. PROD activity of D+R was significantly higher than C, D, D+R+G, D+R+B, and D+R+B+ G. PROD activity of D+B was significantly higher than C and D+R+B. ERND activity of D+R was significantly higher than D+R+G and D+R+B. GST activity of D+R was significantly higher than D+R+G. CAT activity of D+B was significantly lower than C. It is clear that co-administration of resveratrol, berberine, and glibenclamide modifies some of the important xenobiotic metabolizing enzyme activities. Resveratrol and berberine have the potential to cause dietary supplement–drug interaction.

Metabolism of flutamide in diet control fischer 344 and brown Norway x F 344 rats, and its hydroxylation and conjugation by human CYP450s and UDP-glucuronosyltransferases

In our current report, flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl)-phenyl] propanamide) treated diet control and non limited-fed Fischer 344 (F344) and Brown Norway (BN) x F344 rats showed that diet control reduces spontaneous and flutamide-induced hyperplasia((1)). In this continued study, we found that serum concentrations of active metabolite of flutamide, 2-hydroxyflutamide (OH-flu), were 181 ± 26.6 ng/mL and 68 ± 8.0 ng/mL (p<0.05), in non-limited fed and diet control F344 rats. In BN x F344 rats, the serum concentrations of 2-OH-flutamide were 232 ± 57 ng/mL and 52 ± 6 ng/mL (p<0.05) in non-limited fed and diet control animals. In diet control groups, liver microsomes from flutamide-treated F344 rats showed high 7-ethoxyresorufin O-deethylase (EROD) activity, while 7-Benzoxyresorufin O-dealkylase (BROD) activity was not affected significantly. Both rat and human liver microsomes showed flutamide oxidation activity. Human liver microsomes showed 10 times higher activity than rat liver microsomes (0.673 ± 0.04 vs 0.063 ± 0.008 nmol OH-flu/min/mg protein). Microsomes from human tissues such as colon, colon cancer, kidney, bladder, pancreas, prostate, prostate cancer, or ovarian cancer, showed no or non-detectable activity for flutamide hydroxylation. CYP450 1A1, 1A2, 1B1 and 2C19 from human lymphoblastoid cell lines, oxidized flutamide to OH-flu in vitro with activities from 0.118 ± 0.005 to 0.275 ± 0.010 nmol/min/mg protein. Microsomes isolated from human kidney, colon, and liver showed UDP-glucuronosyltransferase (UGT) activity for glucuronidation of OH-flu. Human kidney showed the highest activity. Several human recombinant UGTs (1A1, 1A4, 1A6, 1A7, 1A9, and 1A10) also showed glucuronidation activity for OH-flu. It was found that UGT1A6 was more active than other human UGTs.

Differential responses in the biotransformation systems of the oyster Crassostrea gigas (Thunberg, 1789) elicited by pyrene and fluorene: Molecular, biochemical and histological approach – Part II

Pyrene (PYR) and fluorene (FLU) are among the sixteen priority Polycyclic Aromatic Hydrocarbons (PAH) of the United States Environmental Protection Agency and are both frequently detected in contaminated sites. Due to the importance of bivalve mollusks in biomonitoring programs and the scarce information on the biotransformation system in these organisms, the aim of this study was to investigate the effect of PYR and FLU at the transcriptional level and the enzymatic activities of some biotransformation systems in the Pacific oyster Crassostrea gigas, and to evaluate the histological effects in their soft tissues. Oysters C. gigas were exposed for 24 h and 96 h to PYR (0.25 and 0.5 μM) and FLU (0.6 and 1.2 μM). After exposure, transcript levels of cytochrome P450 coding genes (CYP1-like, CYP2-like, CYP2AU2, CYP356A1, CYP17α-like), glutathione S tranferase genes (omega GSTO-like and microsomal, MGST-like) and sulfotransferase gene (SULT-like), and the activity of ethoxyresorufin O-deethylase (EROD), Glutathione S-transferase (GST) and microssomal GST (MGST) were evaluated in gills. Histologic changes were also evaluated after the exposure period. PYR and FLU bioconcentrated in oyster soft tissues. The half-life time of PYR in water was lower than fluorene, which is in accordance to the higher lipophilicity and bioconcentration of the former. EROD activity was below the limit of detection in all oysters exposed for 96 h to PYR and FLU. The reproductive stage of the oysters exposed to PYR was post-spawn. Exposure to PYR caused tubular atrophy in digestive diverticula, but had no effect on transcript levels of biotransformation genes. However, the organisms exposed for 96 h to PYR 0.5 μM showed higher MGST activity, suggesting a protective role against oxidative stress in gills of oysters under higher levels of PYR in the tissues. Increased number of mucous cells in mantle were observed in oysters exposed to the higher FLU concentration, suggesting a defense mechanisms. Oysters exposed for 24 h to FLU 1.2 μM were in the ripe stage of gonadal development and showed higher transcript levels of CYP2AU2, GSTO-like and SULT-like genes, suggesting a role in the FLU biotransformation. In addition, after 96 h of exposure to FLU there was a significant increase of mucous cells in the mantle of oysters but no effect was observed on the EROD, total GST and MGST activities. These results suggest that PAH have different effects on transcript levels of biotransformation genes and enzyme activities, however these differences could also be related to the reproductive stage.

Integrated assessment of biomarker responses and comparative toxicity pathway in snails Bellamya aeruginosa exposed to tributyltin and triphenyltin in sediments

BackgroundSediments function as a secondary and significant source of tributyltin (TBT) and triphenyltin (TPT) in aquatic ecosystems and may pose a potential threat on benthic organisms and human health. The subchronic toxicity of sediment-associated TBT or TPT to snails Bellamya aeruginosa at environmentally relevant concentrations was investigated in this study. Multiple endpoints at the biochemical [ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), catalase (CAT), protein carbonyl content (PCC) and lipid peroxidation (LPO)] and transcriptomic levels were examined.ResultsTBT or TPT in sediment could induce antioxidant enzymes’ activities and result in oxidative damage in the hepatopancreas of B. aeruginosa after 28-day exposure. A transcriptomic profile of B. aeruginosa exposed to TBT and TPT was reported. CYP genes and EROD activity were sensible and reliable biomarkers for toxicity assessment of TBT or TPT in sediments. Comparative pathway analysis revealed the alteration of steroid hormone biosynthesis and retinol metabolism in B. aeruginosa after 90-day exposure to sediment-associated TBT at the concentration of 2000 ng/g dw, which might affect both reproduction and lipogenesis functions. The ubiquitin proteasome system and immune system might be the toxicity target in B. aeruginosa after exposure to sediment-associated TPT for 90 days.ConclusionsThe results offered new mechanisms underlying the toxicity of sediment-associated tributyltin and triphenyltin.

Toxicological effects of micronized tire crumb rubber on mummichog (Fundulus heteroclitus) and fathead minnow (Pimephales promelas)

Recent studies on the distribution of microplastics in the Charleston Harbor, SC, USA revealed that a large part of the microplastic particles that are found in the intertidal sediments are tire wear particles. These particles originate from the wear of tire treads on roadways, and wash into the estuary during rain events. The abundance of these particles has raised questions about potential toxicity to aquatic organisms that ingest these particles. The synthetic rubber in car tires consists of a large variety of chemicals, which can vary between manufacturers, but usually contains styrene-butadiene rubber, carbon black and zinc. To investigate the potential toxicity of tire wear particles, both mummichogs (Fundulus heteroclitus) and fathead minnow (Pimephales promelas) were exposed to different concentrations of crumb rubber particles (38-355 µm) in a 7-day static renewal exposure. Dissection of the fish revealed that crumb rubber was ingested and accumulated in the intestinal tract. At the highest concentration tested (6 g/L) partial mortality was observed in the fathead minnow, which came close to the assumed LC50. To investigate if polynuclear aromatic hydrocarbons (PAHs) were leaching from the particles, bile fluorescence was measured, together with potential induction of cytochrome P450-1A through the ethoxyresorufin-O-deethylase (EROD) assay. Elevated levels of 2-, 4-, and 5-, ring structures representative of PAHs were detected in the bile of exposed animals. Bile fluorescence indicated that 4-ring PAH compounds were the most bioavailable from the crumb rubber particles. Induction of EROD activity was observed in exposed animals at environmentally relevant concentrations of the crumb rubber particles (<1-2 g/L), and this elevated EROD activity indicated that PAH compounds from the crumb rubber particles were being metabolized in both mummichogs and fathead minnow.

Biomonitoring of aromatic hydrocarbons in clam Meretrix meretrix from an emerging urbanization area, and implications for human health.

Pollution with total petroleum hydrocarbons (TPHs) is a global concern and particularly in coastal environments. Polycyclic aromatic hydrocarbons (PAHs) are regarded as the most toxic components of TPHs and they can also be derived from other sources. Fangcheng Port is considered as a representative emerging coastal city in China, but the status, sources, and hazards to organisms and humans with respect to contamination with PAHs and TPHs are unknown in the coastal regions of this area. Therefore, in this study, we cloned cytochrome P450 family genes (CYP1A1, CYP3A, and CYP4) and heat shock protein 70 gene (HSP70) in the clam Meretrix meretrix as well as optimizing the method for measuring the 7-ethoxyresorufin O-deethylase activity. These molecular indicators and four specific physiological indexes were found to be appropriate biomarkers for indicating the harmful effects of PAHs and TPHs on clams after exposure to the crude oil water-soluble fraction. In field monitoring surveys, we found that the 2- and 3-ring PAHs were dominant in the clams whereas the 4- to 6-ring PAHs were dominant in the sediments at each site. The PAH levels (3.63-12.77ng/g wet weight) in wild clams were lower, whereas the TPH levels (13.25-70.50μg/g wet weight) were higher compared with those determined previous in China and elsewhere. The concentrations of PAHs and TPHs in the sediments (19.20-4215.76ng/g and 3.65-866.40μg/g dry weight) were moderate compared with those in other global regions. Diagnostic ratio analysis demonstrated that the PAHs were derived mainly from pyrogenic sources. The TPHs may have come primarily from industrial effluents, land and maritime transportation, or fishing activities. The Integrated Biomarker Response version 2 indexes indicated that the clams collected from site S5 exhibited the most harmful effects due to contamination by PAHs and TPHs. Human health risk assessments demonstrated that the risks due to PAHs and TPHs following the consumption of clams can be considered acceptable. Our results suggest that continuous monitoring of contamination by PAHs and TPHs is recommended in this emerging coastal city as well as assessing their human health risks.

Pueraria candollei var. mirifica-Induced CYP1A1 and CYP1A2 expression in human choriocarcinoma bewo cells

Background: The human placenta metabolizes many endogenous substances, drugs, and xenobiotics. Cytochrome P450 family 1 (CYP1) is expressed in both early- and full-term placenta. The Thai medicinal plant Pueraria candollei var. mirifica (PM) is traditionally consumed for rejuvenation and has neuroprotective, anti-osteoporotic, and antioxidant activities. Objectives: The objective of this study was to compare the effects of PM and the CYP1A inducer β-naphthoflavone (BNF) on the expression of CYP1, aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), and the transporter ABCG2. Materials and Methods: Human choriocarcinoma BeWo cells were treated with BNF (10 μM), ethanolic extract of PM (PM-EtOH), or column chromatographic fractions of PM-EtOH (F2, F4, and F6) at 1, 10, and 100 μg/mL for 24 h. The mRNA expression of target genes was determined using real-time quantitative polymerase chain reaction. The activity of ethoxyresorufin-O-deethylase (EROD), a marker for CYP1, was measured at the RNA harvesting time point. Results and Discussion: PM-EtOH, F2, and F4 significantly induced EROD activity and expression of CYP1A1 and CYP1A2 while CYP1B1 and AHR were slightly suppressed and ARNT was unchanged. ABCG2 was slightly induced by F2. Therefore, the expression of CYP1 in BeWo cells appears to be independent of the AHR/ARNT regulatory pathway. Conclusion: The use of PM-containing products at high quantities or for long periods during pregnancy is of concern due to likely herb–drug interactions and toxicological risks through activation of CYP1A1 and CYP1A2 transcription.

Screening toxicological effects of different contaminants using hepatic homogenates-based ethoxyresorufin-O-deethylase in vitro

In this paper, we demonstrated the potential of an in vitro method of liver homogenate-based ethoxyresorufin-O-deethylase (EROD) to determine the toxicological effects of multiple kinds of contaminants. We evaluated the in vitro impact of nine pharmaceutically active compounds (PhACs), 13 polycyclic aromatic hydrocarbons (PAHs), and three polychlorinated biphenyls (PCBs). There were different responses of EROD to these contaminants. The response of EROD to PhACs was quite complex, exhibiting both induction and inhibition effects. PAHs and PCBs elicited a strong inhibitory response on EROD activity at high concentrations in a dose-dependent manner. PAHs showed more inhibitory effects as the number of benzene rings increased. Our in vitro bioassay seems to be a potential method for toxicological screening of multiple types of contaminants.

Human CYP1A1 inhibition by flavonoids

Cytochrome P4501A1 (CYP1A1) is involved in the metabolism of several genotoxic/carcinogenic environmental xenobiotics including polycyclic aromatic hydrocarbons (PAHs) like benzo[a]pyrene. Several authors had proposed CYP1A inhibition as a plausible strategy for cancer chemoprevention. Using ethoxyresorufin O-deethylase activity (EROD), we tested the inhibitory properties of nine flavonoids: quercetin, miricetin, luteolin, fisetin, morin, kaempferol, 5-hydroxyflavone (5-HF), 3-hydroxyflavone (3-HF), and flavone (F) against human recombinant CYP1A1. The last three compounds exerted the highest inhibitory effect with IC50 values of 0.07, 0.10 and 0.08 μM respectively; the more hydroxyl-groups were present, the lower the potency of inhibition was. Biochemical characterization leads to the conclusion that flavone and its hydroxy derivatives are mixed-type inhibitors. In silico studies have shown that, Phe224 and other aromatic residues in the human CYP1A1 active site play an important role in flavonoid-CYP interaction, through a π/π stacking between the aminoacid and the flavonoid C-ring. Outside the active site, the three flavonoids bind preferentially between A and K helices of the enzyme. Results from the Ames test using human S9 fraction revealed that none of the three compounds was mutagenic. We can consider 5-HF, 3-HF, and F as potential chemopreventive agents against genotoxic damage caused by metabolites resulting from CYP1A1 activity.

Differential responses in the biotransformation systems of the oyster Crassostrea gasar (Adanson, 1757) elicited by pyrene and fluorene: molecular, biochemical and histological approach - Part I

Polycyclic aromatic hydrocarbons (PAHs) are among the main contaminants in aquatic environments. PAHs can affect organisms due to their carcinogenic, mutagenic and/or teratogenic characteristics. Depending on the PAHs, concentration, and period of exposure, biological damage can occur leading to histopathologic alterations. This study aimed to evaluate the molecular, biochemical and histological responses of the oyster Crassostrea gasar exposed to pyrene (0.25 and 0.5 μM) and fluorene (0.6 and 1.2 μM), after exposure for 24 and 96 h. Concentrations of both PAHs were quantified in the water and in oyster tissues. Transcript levels of phase I (CYP3475C1, CYP2-like, CYP2AU1 and CYP356A) and phase II (GSTO-like, MGST-like and SULT-like) biotransformation-related genes and the activities of ethoxyresorufin-O-deethylase (EROD), total and microsomal glutathione S-transferase (GST and MGST) were evaluated in the gills. Also, histological changes and localization of mRNA transcripts CYP2AU1 in gills, mantle, and digestive diverticula were evaluated. Both PAHs accumulated in oyster tissues. Pyrene half-life in water was significantly lower than fluorene. Transcript levels of all genes were higher in oysters exposed to of pyrene 0.5 μM (24 h). Only CYP2AU1 gene was up-regulated by fluorene exposure. EROD and MGST activities were higher in oysters exposed to pyrene. Tubular atrophy in the digestive diverticula and an increased number of mucous cells in the mantle were observed in oysters exposed to pyrene. CYP2AU1 transcripts were observed in different tissues of pyrene-exposed oysters. A significant correlation was observed between tubular atrophy and the CYP2AU1 hybridization signal in oysters exposed to pyrene, suggesting the sensibility of the species to this PAH. These results suggest an important role of biotransformation-related genes and enzymes and tissue alterations associated to pyrene metabolism but not fluorene. In addition, it reinforces the role of CYP2AU1 gene in the biotransformation process of PAHs in the gills of C. gasar.

Multi-Laboratory Hazard Assessment of Contaminated Microplastic Particles by Means of Enhanced Fish Embryo Test With the Zebrafish (Danio rerio)

As wide-spread pollutants in the marine environment, microplastics (MPs) have raised public concern about potential toxic effects in aquatic organisms, and, among others, MPs were suspected to act as a vector for organic pollutants to biota. The purpose of the present study was to investigate effects by three model pollutants, oxybenzone (BP3), benzo[a]pyrene (BaP) and perfluorooctane sulfonate (PFOS) adsorbed to polyethylene MPs on the basis of a standard assay, the acute fish embryo toxicity test (FET; OECD TG 236) with zebrafish (Danio rerio) supplemented by additional endpoints such as induction of ethoxyresorufin-O-deethylase (EROD) activity, modification of cyp1a gene transcription and changes in larval swimming behavior. FET assays were performed in three laboratories using slightly different husbandry and exposure conditions, which, however, were all fully compatible with the limits defined by OECD TG 236. This allowed for testing of potential changes in the FET assay due to protocol variations. The standard endpoints of the FET (acute embryotoxicity) did not reveal any acute toxicity for both virgin MPs and MPs spiked with BP3, BaP and PFOS. With respect to sublethal endpoints, EROD activity was increased after exposure to MPs spiked with BP3 (3 h pulse) and MPs spiked with BaP (96 h continuous exposure). Cyp1a transcription was increased upon exposure to MPs spiked with BP3 or BaP. For the selected combination of MPs particles and contaminants, the basic FET proved not sensitive enough to reveal effects of (virgin and spiked) MPs. However, given that the FET can easily be supplemented by a broad variety of more subtle and sensitive endpoints, an enhanced FET protocol may provide a relevant approach with developmental stages of a vertebrate animal model, which is not protected by current EU animal welfare legislation (Directive EU 2010/63).

Acute toxic effects caused by the co-exposure of nanoparticles of ZnO and Cu in rainbow trout

The toxic effects produced by the co-exposure to low- and non-toxic concentrations of zinc oxide (ZnONPs) and copper nanoparticles (CuNPs) was assessed in rainbow trout following the OECD Test Guideline 203. Four groups of trouts were exposed for 96 h to a range of concentrations (0.0425–0.34 mg/L) of CuNPs (50 nm) in combination with a fixed non-toxic concentration (1.25 mg/L) of ZnONPs (25 nm) determined from an independent concentration-response study. One additional group was exposed to the highest concentration of CuNPs alone. Behaviour and mortality were observed during the experiment. After 96 h exposure, accumulated levels of Cu and Zn in the fish were measured by ICP-MS and ICP-OES, respectively. The induction of oxidative stress in liver and gills was evaluated by the glutathione-S-transferase (GST) activity and the reduced glutathione (GSH) / oxidized glutathione (GSSG) ratio. The ethoxyresorufin-O-deethylase (EROD) activity was also assessed. The results showed that CuNPs at the highest tested concentration do not cause acute toxicity, whereas exposure to all mixtures caused mortality, which was inversely proportional to the concentration of CuNPs (from 28% to 86% survival). Accumulated levels of Cu and Zn in the fish increased with the increasing concentrations of CuNPs, suggesting that the presence of CuNPs favours the entry of Zn. In general, the GST activity increased significantly in the gills of co-exposed groups, whereas the GSH/GSSG ratio was altered in the liver. The EROD activity was not modified. In conclusion, the co-exposure to these NPs potentiates their toxicity, observing an alteration of the GST activity and GSH/GSSG ratio in gill and liver, which was more pronounced at the lowest concentration of CuNPs. The lower toxic effect observed with the highest concentrations of CuNPs coincides with a greater internalization of Zn.

Liver-specific extracellular matrix hydrogel promotes liver-specific functions of hepatocytes in vitro and survival of transplanted hepatocytes in vivo

A solubilized liver-specific extracellular matrix (L-ECM) substratum was obtained by decellularization of porcine liver using Triton X-100 and pepsin treatments. The L-ECM was able to immobilize hepatocyte growth factor at a high efficiency of 87%. L-ECM gelled spontaneously in a physiologically neutral environment. Primary hepatocytes embedded in the L-ECM gel showed a high albumin synthesis activity and ethoxyresorufin-O-deethylase (EROD) activity even at 3 weeks in culture. In addition, the L-ECM gel-embedded hepatocytes implanted subcutaneously into partial hepatectomized rats showed a high survival rate (18%) and formed a large liver tissue-like structure. Their efficiencies of EROD activity and large liver tissue-like structure formation were about twice those of collagen gel-embedded hepatocytes. Based on these results, we clarified the effectiveness of L-ECM gel as a substrate for hepatocyte culture and transplantation.

Toxicity assessment of pollutants sorbed on environmental sample microplastics collected on beaches: Part I-adverse effects on fish cell line

Microplastics (MPs), are tiny plastic fragments from 1 μm to 5 mm generally found in the aquatic environment which can be easily ingested by organisms and may cause chronic physical but also toxicological effects. Toxicological assays on fish cell lines are commonly used as an alternative tool to provide fast and reliable assessment of the toxic and ecotoxic properties of chemicals or mixtures. Rainbow trout liver cell line (RTLW-1) was used to evaluate the toxicity of pollutants sorbed to MPs sampled in sandy beaches from different islands around the world during the first Race for Water Odyssey in 2015. The collected MPs were analyzed for polymer composition and associated persistent organic pollutants: polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT). In addition, DMSO-extracts from virgin MPs, MPs artificially coated with B[a]P and environmental MPs were analyzed with different bioassays: MTT reduction assay (MTT), ethoxyresorufin-O-deethylase (EROD) assay and comet assay. Microplastics from sand beaches were dominated by polyethylene, followed by polypropylene fragments with variable proportions. Organic pollutants found on plastic from beach sampling was PAHs (2–71 ng g−1). Samples from Bermuda (Somerset Long Bay) and Hawaii (Makapu'u) showed the highest concentration of PAHs and DDT respectively. No toxicity was observed for virgin microplastics. No cytotoxicity was observed on cells exposed to MP extract. However, EROD activity was induced and differently modulated depending on the MPs locations suggesting presence of different pollutants or additives in extract. DNA damage was observed after exposure to four microplastics samples on the six tested. Modification of EROD activity level and DNA damage rate highlight MPs extract toxicity on fish cell line.

Significance of the goby Zosterisessor ophiocephalus as a sentinel species for Venice Lagoon contamination: Combining biomarker responses and bioaccumulation

The Venice Lagoon is an interesting example of an ecosystem suffering for a considerable anthropogenic impact, resulting in high concentrations of persistent organic pollutants (POPs) in lagoon sediments and seafood. In this context, biomonitoring is a crucially important task.The present study aimed at evaluating the validity of a multiple biomarker approach in a benthic fish species. A total of 567 Zosterisessor ophiocephalus (Gobiidae) fish were collected in spring and autumn from three areas of Venice Lagoon (Porto Marghera, Val di Brenta, and Cà Roman) showing high, intermediate and low amounts of POPs, respectively. Aryl hydrocarbon receptor (AHR) and cytochrome P450 1A (CYP1A) mRNA levels, CYP1A protein amount and ethoxyresorufin O-deethylase activity (EROD) were measured in pooled liver and gills (mRNA levels only). Such biological data were then compared with polychlorinated biphenyls (PCBs) residues, measured in grass goby muscle by gas chromatography.Aryl hydrocarbon receptor and CYP1A mRNAs, protein and EROD were upregulated in accordance with PCB amounts measured in Z. ophiocephalus muscles. In fact, the highest AHR and CYP1A induction was observed in fish sampled in close proximity of the industrial area of Porto Marghera.Overall, the present study confirm the grass goby as a reliable sentinel species for Venice Lagoon, and AHR/CYP1A/EROD as a sensitive set of biomarkers of exposure for AHR ligands.

Short-term regulation of the hepatic activities of cytochrome P450 and glutathione S-transferase by nose-only cigarette smoke exposure in mice

The present study aimed to determine the effects of cigarette smoke on the regulation of hepatic cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes in male BALB/c mice exposed to nose-only cigarette smoke for 4 days. There were no significant increases in serum liver injury markers (alanine aminotransferase and aspartate aminotransferase) or oxidative stress (total antioxidant capacity, malondialdehyde, and glutathione disulfide/reduced glutathione) following cigarette smoke exposure, but malondialdehyde was elevated in the bronchoalveolar lavage fluid of smoke-exposed mice. Additionally, the hepatic microsomal protein levels of Cyp1a and Cyp2b, and the activities of ethoxyresorufin O-deethylase, pentoxyresorufin O-depenylase, and chlorzoxazone 6-hydrxylase, were elevated in smoke-exposed mice. Interestingly, the hepatic activities of GST toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, and ethacrynic acid, but not cumene hydroperoxide were enhanced by cigarette smoke exposure, which was consistent with the increased expression levels of mu- and pi-class GSTs, but not alpha-class GSTs, observed in immunoblot analyses. These findings indicate that the short-term inhalation of cigarette smoke induces drug-metabolizing enzymes such as CYP1A, CYP2B, and mu/pi-class GSTs in the absence of hepatic injury and oxidative stress. Furthermore, smoking may alter hepatic drug metabolism, as well as the disposition and toxicity of xenobiotics, including some therapeutic drugs and cigarette smoke constituents.