Hepatic Ethoxyresorufin Research Articles

Reflection of hydrocarbon pollution on hepatic EROD activity in the black goby ( Gobius niger)

Hepatic ethoxyresorufin- O-deethylase (EROD) activity was measured in black gobies ( Gobius niger) collected from seven sampling sites along the Slovenian coast during spawning and non-spawning periods. Sediments from the same sites were tested for their toxicity and for total polycyclic aromatic hydrocarbon (PAH) content. EROD activity was significantly higher during the non-spawning period in winter than during the spawning period in spring and generally correlated well with increased total PAH content in sediments and with sediment toxicity. An 11-fold increase of EROD activity was induced 48 h after injecting 10 μg g −1 weight of benzo(a)pyrene into black gobies. On the basis of these results and of its ecological characteristics, the black goby is proposed as a suitable candidate for future biomonitoring programmes.

Evaluation of the safety and bioactivity of purified and semi-purified glucoraphanin

The anti-carcinogenic effects of broccoli have been attributed to sulforaphane, the hydrolysis product of glucoraphanin (GRP). Here we determined if purified GRP, in the absence of the plant-derived hydrolyzing enzyme myrosinase, could affect pulmonary and hepatic ethoxyresorufin O-deethylase (EROD) and/or NAD(P)H-quinone oxidoreductase 1 (NQO1) activity. Male F344 rats were administered semi-synthetic, semi-purified or purified GRP (240 mg/kg: 550 μmol/kg rat daily for 4 days) by gavage. Hepatic and pulmonary NQO1 activity increased (∼20%), but not EROD. Varying doses of semi-purified GRP (30, 60, or 120 mg/kg rat daily for 4 days) again caused no change in EROD activity, although a dose-dependent increase in NQO1 was seen. Urinary excretion of mercapturic acids showed no difference between preparations, and recovery increased with decreasing dose. Histopathologic examination revealed no abnormal tissues other than cecum, where inflammation was dose dependent; mild at 120 mg/kg and severe at 240 mg/kg, a greatly supra-physiological dose. We conclude that GRP 30–60 mg/kg p.o. is safe and effectively enhances NQO1 in all tissues evaluated.

Chemical contaminants, health indicators, and reproductive biomarker responses in fish from the Colorado River and its tributaries

Common carp (Cyprinus carpio), black bass (Micropterus spp.), and channel catfish (Ictalurus punctatus) were collected from 14 sites in the Colorado River Basin (CRB) to document spatial trends in accumulative contaminants, health indicators, and reproductive biomarkers. Organochlorine residues, 2,3,7,8-tetrachlorodibenzo-p-dioxin-like activity (TCDD-EQ), and elemental contaminants were measured in composite samples of whole fish, grouped by species and gender, from each site. Selenium (Se) and mercury (Hg) concentrations in fish were elevated throughout the CRB, and pesticide concentrations were greatest in fish from agricultural areas in the Lower Colorado River and Gila River. Selenium concentrations exceeded toxicity thresholds for fish (>1.0 microg/g ww) at all CRB sites except the Gila River at Hayden, Arizona. Mercury concentrations were elevated (>0.1 microg/g ww) in fish from the Yampa River at Lay, Colorado; the Green River at Ouray National Wildlife Refuge (NWR), Utah and San Rafael, Utah; the San Juan River at Hogback Diversion, New Mexico; and the Colorado River at Gold Bar Canyon, Utah, Needles, California, and Imperial Dam, Arizona. Concentrations of p,p'-DDE were relatively high in fish from the Gila River at Arlington, Arizona (>1.0 microg/g ww) and Phoenix, Arizona (>0.5 microg/g ww). Concentrations of other formerly used pesticides including toxaphene, total chlordanes, and dieldrin were also greatest at these two sites but did not exceed toxicity thresholds. Currently used pesticides such as Dacthal, endosulfan, gamma-HCH, and methoxychlor were also greatest in fish from the Gila River downstream of Phoenix. Total polychlorinated biphenyls (PCBs; >0.11 microg/g ww) and TCDD-EQs (>5 pg/g ww) exceeded wildlife guidelines in fish from the Gila River at Phoenix. Hepatic ethoxyresorufin O-deethylase (EROD) activity was also relatively high in carp from the Gila River at Phoenix and in bass from the Green River at Ouray NWR. Fish from some sites showed evidence of contaminant exposure as indicated by fish health indicators and reproductive biomarker results. Multiple health indicators including altered body and organ weights and high health assessment index scores may be associated with elevated Se concentrations in fish from the Colorado River at Loma, Colorado and Needles. Although grossly visible external or internal lesions were found on most fish from some sites, histopathological analysis determined many of these to be inflammatory responses associated with parasites. Edema, exophthalmos, and cataracts were noted in fish from sites with elevated Se concentrations. Intersex fish were found at seven of 14 sites and included smallmouth bass (M. dolomieu), largemouth bass (M. salmoides), catfish, and carp and may indicate exposure to endocrine disrupting compounds. A high proportion of smallmouth bass from the Yampa River at Lay (70%) was intersex but the cause of this condition is unknown. Male carp, bass, and catfish with low concentrations of vitellogenin were common in the CRB. Comparatively high vitellogenin concentrations (>0.2 mg/mL) were measured in male bass from the Green River at Ouray NWR and the Colorado River at Imperial Dam and indicate exposure to estrogenic or anti-androgenic chemicals. Anomalous reproductive biomarkers including low GSI and gonadal abnormalities (calcifications, edema, and parasites) observed in fish downstream of Phoenix are likely related to the poor water-quality of the Gila River in this area.

Biomarkers of Contaminant Exposure in Northern Pike (Esox lucius) from the Yukon River Basin, Alaska

As part of a larger investigation, northern pike (n = 158; Esox lucius) were collected from ten sites in the Yukon River Basin (YRB), Alaska, to document biomarkers and their correlations with organochlorine pesticide (total p,p'-DDT, total chlordane, dieldrin, and toxaphene), total polychlorinated biphenyls (PCBs), and elemental contaminant (arsenic, cadmium, copper, lead, total mercury, selenium, and zinc) concentrations. A suite of biomarkers including somatic indices, hepatic 7-ethoxyresorufin O-deethylase (EROD) activity, vitellogenin concentrations, steroid hormone (17B- ustradiol and 16-kebtestosteront) concentrations, splenic macrophage aggregates (MAs), oocyte atresia, and other microscopic anomalies in various tissues were documented in YRB pike. Mean condition factor (0.50 to 0.68), hepatosomatic index (1.00% to 3.56%), and splenosomatic index (0.09% to 0.18%) were not anomalous at any site nor correlated with any contaminant concentration. Mean EROD activity (0.71 to 17.51 pmol/min/mg protein) was similar to basal activity levels previously measured in pike and was positively correlated with selenium concentrations (r = 0.88, P < 0.01). Vitellogenin concentrations in female (0.09 to 5.32 mg/mL) and male (<0.0005 to 0.097 mg/mL) pike were not correlated with any contaminant, but vitellogenin concentrations >0.01 mg/mL in male pike from multiple sites indicated exposure to estrogenic compounds. Mean steroid hormone concentrations and percent oocyte atresia were not anomalous in pike from any YRB site. Few site differences were significant for mean MA density (1.86 to 6.42 MA/mm(2)), size (812 to 1481 microm(2)), and tissue occupied (MA-%; 0.24% to 0.75%). A linear regression between MA-% and total PCBs was significant, although PCB concentrations were generally low in YRB pike (< or =63 ng/g), and MA-% values in female pike (0.24% to 0.54%) were lower than in male pike (0.32% to 0.75%) at similar PCB concentrations. Greater numbers of MAs were found as zinc concentrations increased in YRB female pike, but it is unlikely that this is a causative relationship. Histological abnormalities observed in gill, liver, spleen, and kidney tissues were not likely a result of contaminant exposure but provide information on the general health of YRB pike. The most common histologic anomalies were parasitic infestations in various organs and developing nephrons and nephrocalcinosis in posterior kidney tissues. Overall, few biomarker responses in YRB pike were correlated with chemical contaminant concentrations, and YRB pike generally appeared to be healthy with no site having multiple anomalous biomarker responses.

Multiscale approach of fish responses to different types of environmental contaminations: A case study

In order to study the responses of wild fish to environmental contaminations, different sites (two references and three contaminated) were sampled across the Walloon hydrographical network (southern Belgium). The status of fish communities was characterized according to an index of biotic integrity (IBI). Furthermore, population structure, reproductive parameters and biochemical assays were performed on chub ( Leuciscus cephalus) as sentinel species. The results showed impaired responses in fish sampled downstream paper mill effluents (BKME), in a polychlorinated biphenyls (PCB) contaminated site, and in a river receiving pesticides (i.e. atrazine and hexachlorocyclohexane). High level of hepatic 7-ethoxyresorufin- Odeethylase (EROD) activity were evidence of direct toxicant exposure, while population structures showed unbalanced size–frequency distributions. Moreover, in the pesticide contaminated site, a higher proportion of atretic oocytes was found in females and abnormally high plasma alkali–labile phosphorus (ALP) values were noticed in males, compared to reference sites, suggesting that chubs were exposed to estrogenic compounds. IBI scores from both sites were also lower than in reference sites. On the other hand, no particular response was found in hepatic glutathione- S-transferase (GST) activities, hepatic heat-shock protein HSP70 concentrations and plasma sex steroids (estradiol-17β, 11-ketotestosterone and testosterone) in chubs from any sites. By using a multiparametric approach, a better understanding of response mechanisms to pollution in fish may be achieved in order to improve the ecological status of river ecosystems.

Effects of pentachlorophenol on the reproduction of Japanese medaka ( Oryzias latipes)

Pentachlorophenol (PCP) is widely used to control termites and protect wood from fungal-rot and wood-boring insects, and is often detected in the aquatic environment. Few studies have evaluated PCP as an environmental endocrine disruptor. In the present work, Japanese medaka ( Oryzias latipes) was exposed to PCP for 28 days (F0 generation) with subsequent measurements of vitellogenin (VTG), hepatic 7-ethoxyresorufin- O-deethylase (EROD), and reproductive endpoints. Plasma VTG significantly increased in male fish treated with PCP concentrations lower than 200 μg/l and decreased in male and female animals exposed to 200 μg/l. Hepatic EROD from female fish increased when PCP exposure concentrations exceeded 20 μg/l, but decreased in the 200 μg/l PCP treatment group. Fecundity and mean fertility of female medaka decreased significantly in the second and third week following exposure concentrations greater than 100 μg/l, and testis-ova of male medaka was observed at PCP concentrations greater than 50 μg/l. Histological lesions of liver and kidney occurred when exposure concentrations exceeded 50 μg/l. In F1 generations, the hatching rates and time to hatch of offspring were significantly affected in fish exposed to 200 μg/l. These results indicated that PCP exposure caused responses consistent with estrogen and aryl hydrocarbon receptor activation as well as reproductive impairment at environmentally relevant concentrations.

Enhancement of Dioxin Toxicity with an Anti-Stress Drug, Carbenoxolone, in Mice

The effect of the carbenoxolone (CBX) on the subacute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in C57BL/6J mice. The loss of body weight due to TCDD was enhanced by simultaneous treatment with CBX. In agreement with the change in body weight, CBX failed to improve TCDD-induced hepatic hypertrophy and thymic atrophy. Co-treatment of CBX with TCDD tended to cause hepatic hypertrophy more extensively than did TCDD alone, and combined treatment induced significant renal hypertrophy and splenic atrophy which were not seen in mice treated with TCDD or CBX alone. CBX had no effect on the TCDD-mediated induction of hepatic ethoxyresorufin O-deethylase activity, a marker for aryl hydrocarbon receptor (AhR)-linked gene expression. These results suggest that CBX enhances TCDD toxicity by a mechanism(s) distinct from activation of the AhR.

Low hepatic ethoxyresorufin‐o‐deethylase activity correlates with high organochlorine concentrations in Atlantic tomcod from the Canadian east coast

A suppression of hepatic ethoxyresorufin-O-deethylase (EROD) activity was reported recently in large-sized Atlantic tomcod (Microgadus tomcod) from the St. Lawrence Estuary (SLE; QC, Canada), possibly related to chronic exposure to persistent contaminants and/or to emaciation. In the present study, hepatic concentrations of organochlorine contaminants and biological responses were measured in female tomcods from three estuaries located on the Canadian east coast: The SLE, the Miramichi (ME), and the Richibucto (RE) Rivers Estuaries (NB, Canada). Tomcods from the SLE had higher hepatic concentrations of organochlorine contaminants than tomcods from the ME and RE. For example, concentrations of polychlorinated biphenyls (PCBs, lipid wt) were 2.5 to 4 times higher, and concentrations of mirex and chlordanes were 6 times higher, in tomcods from the SLE than in tomcods from the other sites. Concentrations of polycyclic aromatic hydrocarbons (PAHs) metabolites in the bile did not differ among sites. The pattern of biological responses differed markedly between the SLE and the two other sites. Tomcods from the SLE had 1.5 times higher concentrations of DNA adducts and 2 times higher rates of hepatocellular proliferation, but 20 times lower hepatic EROD activity, than tomcods from the ME and RE. Lipid content was not correlated with EROD activity, indicating that low hepatic lipid content alone does not cause suppression of EROD activity in Atlantic tomcod. In contrast, for the three sites combined, EROD activity decreased as concentrations of PCBs increased. Within sites, hepatic PCB concentrations increased as lipid content decreased. This study supports the hypothesis that low EROD activity in SLE tomcods is related to chronic exposure to organochlorine contaminants.

Rubber additives induce oxidative stress in rainbow trout

Several studies have demonstrated the toxicity of rubber leachate, mainly from rubber tires, to aquatic organisms. In the present study rainbow trout ( Oncorhynchus mykiss) were exposed to water provided to aquaria through a rubber hose. Increased hepatic ethoxyresorufin- O-deethylase (EROD) activity, and glutathione reductase (GR) activity were observed in the exposed fish. Two common rubber additives, 2-mercaptobenzothiazole (MBT) and diphenylamine (DPA) and structurally related compounds, were identified by chemical analyses of water samples as were hydroxylated polycyclic aromatic hydrocarbons. Metabolites of these compounds were also detected in the bile of exposed fish, as were some of the parent compounds. In a following experiment , we injected rainbow trout with DPA or MBT. Both compounds affected total glutathione (tGSH) concentration in liver and MBT caused an increase in hepatic GR and glutathione S-transferase (GST) activity as well. In DPA injected fish, hydroxylated DPA was the main metabolite in the bile. Our results indicate that rubber chemicals may leach into the water surroundings where they can be taken up and metabolised by fish. Some of these chemicals can lead to up-regulation of antioxidant defences as demonstrated with DPA and MBT injections.

Summary results from a pilot study conducted around an oil production platform on the Northwest Shelf of Australia

The Australian Institute of Marine Science (AIMS) conducted a pilot study around the Harriet A oil production platform on the Northwest Shelf of Australia. We evaluated hepatic ethoxyresorufin- O-deethylase (EROD) activity, fluorescent aromatic compounds (FACs) in bile and immunodetection of CYP1A-like proteins in two Australian tropical fish species, Gold-Spotted Trevally ( Carangoides fulvoguttatus) and Bar-Cheeked Coral Trout ( Plectropomus maculatus) to assess exposure to petroleum hydrocarbons associated with produced formation water (PFW). Additionally, the incidence of hydrocarbon-degrading bacteria isolated from the liver and bile of all fish captured was examined. Low EROD activity was found in both species, with EROD activity in C. fulvoguttatus showing significant site differences. FACs and CYP1A protein levels in C. fulvoguttatus showed a clear trend in hydrocarbon exposure consistent with hydrocarbon chemistry data: Harriet A > Harriet C > reference site. P. maculatus showed elevated levels of FACs at Harriet A as compared to the reference site and demonstrated detectable levels of CYP1A-like proteins at these two sites. Hydrocarbon-degrading bacteria were found in the liver and bile of both species, yet there was no correlation by sites. Our results demonstrate that C. fulvoguttatus and P. maculatus have potential as indicator species for assessing the effects from exposure to petroleum hydrocarbons. Both FACs and CYP1A are providing warning signs that there is potential for biological effects on fish populations exposed to PFW around the Harriet A production platform.

Environmental contaminants and biomarker responses in fish from the Rio Grande and its U.S. tributaries: Spatial and temporal trends

We collected, examined, and analyzed 368 fish of seven species from 10 sites on rivers of the Rio Grande Basin (RGB) during late 1997 and early 1998 to document temporal and geographic trends in the concentrations of accumulative contaminants and to assess contaminant effects on the fish. Sites were located on the mainstem of the Rio Grande and on the Arroyo Colorado and Pecos River in Texas (TX), New Mexico (NM), and Colorado. Common carp ( Cyprinus carpio) and largemouth bass ( Micropterus salmoides) were the targeted species. Fish were examined in the field for internal and external visible gross lesions, selected organs were weighed to compute ponderal and organosomatic indices, and samples of tissues and fluids were obtained and preserved for analysis of fish health and reproductive biomarkers. Whole fish from each station were composited by species and gender and analyzed for organochlorine chemical residues and elemental contaminants using instrumental methods, and for 2,3,7,8-tetrachloro dibenzo- p-dioxin-like activity (TCDD-EQ) using the H4IIE rat hepatoma cell bioassay. Overall, fish from lower RGB stations contained greater concentrations of organochlorine pesticide residues and appeared to be less healthy than those from sites in the central and upper parts of the basin, as indicated by a general gradient of residue concentrations and biomarker responses. A minimal number of altered biomarkers and few or no elevated contaminant concentrations were noted in fish from the upper RGB. The exception was elevated concentrations [up to 0.46 μg/g wet-weight (ww)] of total mercury (Hg) in predatory species from the Rio Grande at Elephant Butte Reservoir, NM, a condition documented in previous studies. Arsenic (As) and selenium (Se) concentrations were greatest in fish from sites in the central RGB; Se concentrations in fish from the Pecos River at Red Bluff Lake, TX and from the Rio Grande at Langtry, TX and Amistad International Reservoir, TX exceeded published fish and wildlife toxicity thresholds. In the lower RGB, residues of p,p′-DDT metabolites (≤1.69 μg/g ww), chlordane-related compounds (≤0.21 μg/g ww), dieldrin (≤0.0.05 μg/g ww), and toxaphene (≤2.4 μg/g ww) were detected in fish from most sites; maximum concentrations were in channel catfish ( Ictalurus punctatus) from the Arroyo Colorado at Harlingen, TX. Concentrations of one or more residues exceeded toxicity thresholds for fish and wildlife in fish from this site and from the Rio Grande at Mission, TX and Brownsville, TX; however, concentrations were lower than those reported by previous studies. In addition, the proportional concentrations of p,p′-DDT at all sites were low, indicating weathered DDT rather than the influx of new material. Concentrations of total PCBs (<0.05 μg/g ww) and TCDD-EQ (≤6 pg/g ww) were comparatively low in all samples. Hepatic ethoxyresorufin O-deethylase (EROD) activity in some fish was elevated relative to reference rates at most sites, but was generally lower than previously reported activity in fish from heavily contaminated locations. The comparatively low PCB and TCDD-EQ concentrations together with elevated EROD activity may reflect exposure to polycyclic aromatic hydrocarbons. Reproductive biomarkers were consistent with chronic contaminant exposure at lower RGB sites; comparatively large percentages of intersex male largemouth bass, relatively low gonadosomatic indices, and elevated plasma vitellogenin concentrations in male fish were noted at three of the four stations. Large percentages of atretic eggs were also observed in the ovaries of female common carp from the Rio Grande at Brownsville, TX. Although many of the conditions noted may have other causes in addition to contaminant exposure, the biomarker results for the lower RGB sites are consistent with subtle responses of fish to contaminants, an interpretation supported by the chemical data of this and other investigations.

Effects of Postnatal Exposure to a Mixture of Polychlorinated Biphenyls, p,p′-dichlorodiphenyltrichloroethane, and p-p′-dichlorodiphenyldichloroethene in Prepubertal and Adult Female Sprague-Dawley Rats

The postnatal period is a critical phase of development and a time during which humans are exposed to higher levels of persistent organic pollutants (POPs), than during subsequent periods of life. There is a paucity of information describing effects of postnatal exposure to environmentally relevant mixtures of POPs, such as polychlorinated biphenyls (PCBs), p,p'-dichlorodiphenyltrichloroethane (DDT), and p,p'-dichlorodiphenyldichloroethene (DDE). To provide data useful for the risk assessment of postnatal exposure to POPs, mixtures containing 19 PCBs, DDT, and DDE were prepared according to their concentrations previously measured in the milk of Canadian women, and dose-response effects were tested on the proliferation of MCF7-E3 cells in vitro, and in vivo experiments. Female neonates were exposed by gavage at postnatal days (PNDs) 1, 5, 10, 15, and 20 with dosages equivalent to 10, 100, and 1000 times the estimated human exposure level over the first 24 days of life. The MCF7-E3 cells showed a 227% increase in the AlamarBlue proliferation index, suggesting estrogen-like properties of the mixture, but this was not confirmed in vivo, given the absence of uterotrophic effects at PND21. An increase (511%) in hepatic ethoxyresorufin-o-deethylase activity at the dose 100 x was the most sensitive endpoint among those measured at PND21 (organ weight, mammary gland and ovarian morphometry, hepatic enzyme inductions, serum thyroxine and pituitary hormones). In liver samples from older female rats (previously involved in a mammary tumor study [Desaulniers et al., Toxicol. Sci. 75:468-480, 2001]), hepatic metabolism of 14C-estradiol-17beta (E2) at PND55 to PND62 was significantly higher in the 1000x compared to the control group, but hepatic detoxification enzyme activities had already returned to control values. The production of hepatic 2-hydroxy-E2 decreased, whereas that of estrone increased with age. In conclusion, the smallest dose of the mixture to induce significant effects was 100x, and mixture-induced changes in the hepatic metabolism of estrogens might be a sensitive indicator of persistent effects.

A Bio-Effect Directed Fractionation Study for Toxicological and Chemical Characterization of Organic Compounds in Bottom Sediment

The major aim of this study was to characterize toxic organic compounds in bottom sediments from a PCB polluted bay. To overcome difficulties in pinpointing toxicants in complex environmental samples we applied a bio-effect directed (BED) fractionation approach and investigated the relationships between aromaticity, teratogenicity, and aryl hydrocarbon receptor (AhR) mediated toxicity. Hepatic ethoxyresorufin O-deethylase (EROD) activities and malformations were investigated in rainbow trout (Oncorhynchus mykiss) larvae exposed by injecting sediment extract and fractions (separated by their degree of aromaticity) thereof into newly fertilized eggs. Our results imply that non-additive effects get more pronounced the more complex the exposure. The fraction mainly composed of dicyclic aromatic compounds (DACs), including PCBs, was surprisingly less teratogenic than the fraction mainly composed of polycyclic aromatic compounds (PACs). A major part of the latter potential was isolated in a subfraction mainly composed of three- and four-ring compounds (including alkylated and sulphur-heterocyclic compounds). Though no clear relationship between aromaticity and EROD induction was observed, both the DAC- and the PAC-fractions contributed equally to the EROD induction potential. A major part of the PAC-fraction's induction potential came from a subfraction containing compounds with more than five rings. No clear relationship between teratogenicity and EROD induction was observed, underlining the need for a battery of biomarkers in estimating environmental risk. Two specific malformations not previously described in literature-asymmetric yolk sac and fin edema-could be tracked through the fractionation steps, suggesting that this BED-fractionation strategy is a reliable tool for pinpointing toxic compounds in the environment.

Contribution of commonly analyzed polycyclic aromatic hydrocarbons (PAHs) to potential toxicity in early life stages of rainbow trout (Oncorhynchus mykiss)

In a series of bio-effect-directed fractionation experiments, we investigated the potential toxicity of sediment extracts from a contaminated bay. A previous study investigated abnormalities and hepatic ethoxyresorufin O-deethylase (EROD) activities in rainbow trout (Oncorhynchus mykiss) larvae by exposing newly fertilized eggs to the total extract and to fractions separated by degree of aromaticity. A major part of the potential toxicity was isolated in a fraction containing polycyclic aromatic compounds (PACs). In this study, we prepared a synthetic PAC mixture with 17 commonly analyzed polycyclic aromatic hydrocarbons (PAHs) in amounts equimolar to those found in the sediment PAC fraction. The 17 PAHs, which included 11 of the 16 United States Environmental Protection Agency (US EPA) priority PAHs, were unable to account for the toxicopathic effects observed and could explain less than 4% of the total EROD induction. The lack of a clear relationship between toxicopathic effects and EROD induction underlines the need for a battery of biomarkers for estimating environmental risk. These results reveal the limits of our knowledge regarding compounds responsible for potential toxicity in field situations.

Beta-naphthoflavone inhibits the induction of hepatic oestrogen-dependent proteins by 17alpha-ethynylestradiol in mosquitofish (Gambusia holbrooki)

The interactive effects of an aryl hydrocarbon receptor (AhR) agonist and of a xenoestrogen on biomarker responses were studied in the liver of male mosquitofish (Gambusia holbrooki). Hepatic 7-ethoxyresorufin O-deethylase (EROD) enzymatic activity was measured as a biomarker of exposure to the model AhR agonist beta-naphthoflavone (bNF). Hepatic proteins indicating the exposure of males to the synthetic oestrogen 17alpha-ethynylestradiol (EE2) were monitored by Western blot analysis using immunoserum prepared for this study. After a semi-static exposure only to waterborne EE2, Western blot analysis of liver homogenate revealed the induction of two protein bands (a double band at 205 kDa and a single band at 125 kDa). The interaction between bNF and EE2 was investigated by analysing, on the one hand, EROD activity and, on the other hand, immunoreactivity corresponding to the two oestrogen-dependent protein bands in the liver of fish exposed to different concentrations of bNF for 2 days, then to the same concentrations of bNF plus 0.1 µg l−1 EE2 for 5 days. EE2 changed neither the basal activity of EROD nor its rate of induction with 1.0 and 4.0 µg l−1 bNF. On the other hand, the induction of oestrogen-dependent proteins with 0.1 µg l−1 EE2 was inhibited by exposure to 4.0 µg l−1 bNF. These results together with literature data suggest that field monitoring of xenoestrogen contamination through the analysis of oestrogen-dependent protein in male fish as a biomarker should take into account the possible negative interference of AhR agonists.

Organochlorine contaminants in flounder ( Platichthys flesus) and mullet ( Mugil cephalus) from Douro estuary, and their use as sentinel species for environmental monitoring

In order to monitor the presence of organic pollutants in Douro estuary (NW Portugal), two sentinel fish species, grey mullet ( Mugil cephalus) and flounder ( Platichthys flesus), were periodically sampled from May 2001 to April 2002. At all sampling periods, several specimens of both species were allowed to depurate in clean sea water. Levels of PCBs and DDTs were quantified in liver and muscle of both species. The accumulation of PCBs was higher in muscle of mullet, with a maximum of 345 ng/g dw, than in flounder, with a maximum 52 ng/g dw. In the liver, flounder showed the highest levels (811 ng/g dw). Of the 18 congeners analysed, CBs 180 (hepta), 153 and 138 (hexachlorobiphenyls) were predominant in the tissue of both species. The maximum concentration of tDDT was measured in flounder liver (301 ng/g dw). In contrast, a 10-fold higher tDDT was recorded in mullet muscle (63–69 ng/g dw and 8–16 ng/g dw for mullet and flounder, respectively). The hepatic ethoxyresorufin O-deethylase (EROD) activity was periodically determined. Consistently, a 10-fold higher enzymatic activity was present in mullet in comparison with flounder (1536 pmol/min /mg protein in mullet and 156 pmol/min /mg protein in flounder). In mullet, no correlation could be found between EROD activity and gonado-somatic index (GSI) or hepato-somatic index (HSI). On the contrary, during the reproductive season, female flounder showed a negative correlation between EROD activity and GSI. Despite being in clean sea water for 1 month period, no significant decrease in the tissue content of PCBs and tDDT was found. However, mullet’s EROD activity followed a clear pattern, with a decrease enzymatic activity after being in captivity. Female flounder displayed a similar trend during the resting season. Yet, during the reproductive season, an increased EROD activity was recorded after being in captivity for 1 month, which may be associated with a modulation effect of steroids on CYP 1A1. Histological analyses of gonad revealed that 21% of male mullet displayed testis-ova, while no male flounder was found to show gonadal abnormalities. Overall, the study reports the accumulation of significant levels of PCBs and DDTs in the tissues of the studied species. It also provides important evidences supporting the use of grey mullet as a sentinel species for monitoring the presence of organic contaminants and xeno-estrogenic pollution in southern European estuaries.

Cytological changes in association with ethoxyresorufin O‐deethylase induction in fish upon dietary exposure to benzo[a]pyrene

Juvenile areolated grouper (Epinephelus areolatus) were exposed to two levels of dietary benzo[a]pyrene (BaP; 0.25-12.5 microg/g body wt/d) for four weeks, followed by four weeks of depuration. Significant increase in hepatic ethoxyresorufin O-deethylase (EROD) activities was found after one week, preceding an increase in lipopigments (as measured by quantitative transmission electron microscopy) in week 2 of exposure. The EROD activities in the BaP-treated fish subsided at week 4 of exposure and throughout the depuration period. Lipopigments in the high-dose group appeared to be more persistent than that of the EROD activity during the exposure period and remained significantly higher than that of the controls at week 4. Levels of lipopigments, however, rapidly subsided on withdrawal of BaP exposure. These results appear to suggest that changes in EROD activities would precede cytological changes and that both the observed cytological and biochemical changes are reversible. Results of the present study also lend further support to our earlier findings on Solea ovata, that a significant relationship exists between EROD activity and lipopigment accumulation (as measured by volume density, absolute volume, numerical density, and absolute density; r = 0.483-0.358, p < 0.05), regardless of fish species (S. ovata and aerolated grouper) as well as the routes of exposure to BaP (intraperitoneal injection or dietary exposure). This provides strong supporting evidence that elevated EROD activities in fish liver do not merely indicate exposure to polyaromatic hydrocarbons (PAHs) but are also associated with significant biological effects. Our results showed that hepatic EROD activity and lipopigments could be used to indicate recent exposure of the fish to BaP/PAHs.

β-Naphthoflavone alters normal plasma levels of vitellogenin, 17β-estradiol and luteinizing hormone in sea bass broodstock

A variety of environmental pollutants exhibit antiestrogenic properties, i.e. these compounds antagonize estrogen-dependent processes in their target tissues. This is the case with β-naphthoflavone (βNF), a compound that is known for its ability to bind to the arylhydrocarbon receptor (AhR) and thereby to induce cytochrome P4501A (CYP1A) expression. In previous studies, we have shown that βNF antagonizes the 17β-estradiol (E2)-mediated production of vitellogenin (VTG) in cultured rainbow trout hepatocytes in vitro. This antiestrogenic effect appeared to be related to the βNF activation of the AhR pathway. The questions addressed in the present study are: (a) if βNF is able to evoke an antiestrogenic effect in fish in vivo; this was estimated from circulating VTG levels, and (b) if the antiestrogenic action is expressed not only at the cellular (suppressed VTG synthesis) but also at the systemic level; this was addressed by measuring the circulating level of two hormones directly implicated in the regulation of reproductive processes: E2 and luteinizing hormone (LH). As experimental model, we used broodstock sea bass ( Dicentrarchus labrax). The fish were fed with a βNF-enriched diet from December, at the beginning of the reproductive period of sea bass, until the termination of this period in April. Induction of hepatic 7-ethoxyresorufin- O-deethylase (EROD) activity and CYP1A mRNA levels in βNF-fed fish indicates that the test compound was accumulated and effective. Compared to the control fish, βNF treatment of sea bass was associated with a significant reduction of plasma VTG levels, and it disturbed the reproduction-related fluctuations of plasma E2 and LH levels. These findings point to an antiestrogenic effect of βNF on VTG synthesis and to an interference with the hypothalamus-pituitary-gonad axis in reproducing sea bass.

Hepatic ethoxyresorufin O-deethylase (EROD) activity in flounder ( Platichthys flesus) from contaminant impacted estuaries of the United Kingdom: continued monitoring 1999–2001

The determination of hepatic ethoxyresorufin O-deethylase (EROD) has been used to assess the induction of the mixed function oxygenase system (MFO) of flounder ( Platichthys flesus) in UK estuaries. Induction of the MFO system denotes possible exposure to certain organic contaminants (e.g. polycyclic aromatic hydrocarbons, polychlorinated biphenyls) and its measure has been incorporated in national monitoring programmes. This study presents EROD monitoring data from 5 UK estuaries taken between 1999 and 2001 and builds on data from previous years. The results reveal that for all sampled estuaries EROD values have been significantly ( p<0.05) elevated on the majority of occasions in comparison with the reference estuary, the Alde in Suffolk, UK. However, the limited temporal scale of the reported monitoring does not allow any conclusions to be drawn with respect to trends in the data. Possible factors influencing the data (size, gender, seasonality, reproductive status, etc.) are discussed and recommendations for continued monitoring are made.

Reduction of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice using an antiulcer drug, geranylgeranylacetone.

The protective effect of geranylgeranylacetone (GGA), an antiulcer drug, against the acute toxicity and teratogenicity produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined in C57BL/6J mice. When mice were co-treated, GGA reduced the loss of body weight gain and lethality produced by TCDD but hepatomegaly and thymic atrophy were not improved. Additionally, no protective effect of GGA was observed in the formation of cleft palate and hydronephrosis in mouse fetuses caused by maternal exposure to TCDD. To clarify the reducing mechanism by GGA, the Hsp70.1 mRNA levels in liver and intestine were analyzed. However, it was difficult to explain the effect of GGA from the induction of Hsp70.1. GGA had also no effect on the induction of hepatic ethoxyresorufin O-deethylase activity by TCDD. These data suggest that GGA exhibits a protective effect against some forms of dioxin toxicity by a mechanism without involving inhibition of arylhydrocarbon receptor activation.