Augustine Arukwe

Induction of hepatic estrogen receptor in juvenile Atlantic salmon in vivo by the environmental estrogen, 4-nonylphenol

Alkylphenol ethoxylate degradation products such as nonylphenol and octylphenol are shown to have estrogenic effects. Nonylphenol induces synthesis of vitellogenin (a precursor of egg yolk proteins) and zona radiata proteins (eggshell proteins) in juvenile and/or male fish. Little is known about the molecular mechanisms of estrogenicity of environmental chemicals such as nonylphenol. To study the mechanisms of estrogenic effects of 4-nonylphenol (NP), we examined its in vivo effects on the expression of the estrogen receptor (ER), vitellogenin (Vtg) and zona radiata protein (Zrp) genes in juvenile Atlantic salmon liver. We show that the ER mRNA synthesis is induced by NP in a dose-dependent manner in juvenile Atlantic salmon liver. The induction of the ER mRNA synthesis is followed by the induction of Zrp and Vtg mRNA synthesis. The ER transcripts reach peak levels earlier than the Zrp and Vtg mRNA and proteins, which is in agreement with the physiological effects of estradiol during zonagenesis and vitellogenesis. Various studies have also shown that NP competitively inhibits the binding of 17 beta-estradiol (E2) to ER. Our results further suggest that NP directly mimics E2 in inducing the ER, Zrp and Vtg genes in salmon liver.

Differential biomarker gene and protein expressions in nonylphenol and estradiol-17β treated juvenile rainbow trout (Oncorhynchus mykiss)

The time- and dose-dependent transcriptional and translational expression of biomarker genes in nonylphenol (NP) and estradiol-17beta (E(2)) treated juvenile rainbow trout is reported. Fish were exposed to NP (1, 5 and 25 mg/kg) and E(2) (5 mg/kg) and killed at 2, 6, 12, 24, 48 and 72 h after exposure. The estrogen receptor (ER), vitellogenin (Vtg) and eggshell zona radiata protein (Zr-protein) gene expressions were analyzed in total liver RNA using Northern and slot hybridization with specific cDNA probes. Plasma Vtg and Zr-protein levels were evaluated using indirect ELISA. While Zr-protein gene showed an induction only at 24 h post-exposure, the plasma protein levels showed a time-dependent increase in the 25-mg NP treated group. Vtg transcripts showed an apparent time-dependent increase without a concomitant increase in protein levels in the 25-mg NP treated fish. Time-dependent increases in Vtg and Zr-protein gene expressions without the corresponding increases in ER gene transcription was observed in E(2)-treated fish at 2, 6 and 12 h post-exposure. Induction of ER gene transcripts was observed from 24 h and did not change significantly at 48 and 72 h. In the E(2)-treated fish, induction of plasma Vtg levels was observed at 48 and 72 h, while plasma Zr-protein was induced at 24, 48 and 72 h, after exposure. We conclude that the E(2)- and NP-induced Vtg and Zr-protein gene expressions at the early time intervals after exposure are not dependent on increase in the transcriptional activity of the ER gene and that Vtg and Zr-protein gene transcriptions require only basal or minimal ER concentration, in addition to other mechanisms.

In vivo and in vitro metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar)

In the environment, nonylphenol (NP) occurs predominantly as a degradation product of nonylphenol ethoxylate (NPE). They can be found in many types of products including detergents, plastics, emulsifiers, pesticides, and industrial and consumer cleaning products. As a consequence of their use in a variety of products, they are quite common in rivers and other aquatic environments that receive sewage discharges. Because of its enhanced resistance towards biodegradation, toxicity, estrogenic effects, and ability to bioaccumulate in aquatic organisms NP has been regarded as the most critical metabolite of APEs. We have studied the in vivo and in vitro metabolism and organ distribution of NP in juvenile salmon. Fish were exposed in vivo to waterborne [3H]-4-n-NP for a period up to 72 h or were administered a single oral dose of [3H]-4-n-NP. In vitro biotransformation of NP was studied by exposure of cultured salmon hepatocytes to [3H]-4-n-NP in the presence or absence of a CYP1A-inducer, beta-naphthoflavone (betaNF). Our results show that 4-n-NP was mainly metabolized in vivo, to its corresponding glucuronide conjugates and hydroxylates. The major route of excretion was the bile. The half-life of residues in carcass and muscle was between 24 and 48 h in both waterborne and dietary exposure. In whole body autoradiography, intragastric administered [3H]-4-n-NP was mainly present in the gastrointestinal tract and bile. NP-derived radioactivity in fish exposed via water was more evenly distributed in the organs compared to intragastric exposure and were observed in the intestinal contents, liver, kidney, gills, skin, abdominal fat and brain. In vitro pretreatment of hepatocytes with betaNF had no effect on rates or patterns of NP biotransformation. The in vitro metabolic rate of NP were 118 pmol NP metabolized/h/0.5x10(6) cells without betaNF, and 98 pmol NP metabolized/h/0.5x10(6) cells when betaNF was added to the culture medium.

Cellular and Molecular Responses to Endocrine-Modulators and the Impact on Fish Reproduction

Anthropogenic chemicals in the aquatic environment are known to cause reproductive disturbances in vertebrate and invertebrate organisms, by interfering with the endocrine systems. Laboratory-based in vivo and in vitro studies have indicated that several of the anthropogenic and other naturally occurring chemicals in the environment can cause adverse reproductive effects. Various definite or possible reproductive abnormalities caused by endocrine disruption have been identified, but in majority of the reported cases, it is not known whether adverse effects have occurred in the population level of biological organization. Disruption of the hormonal functions in fish may have effects on a number of events, including sexual maturation, gamete production and transport, sexual behaviour, fertility, gestation, lactation or modifications in other functions that are dependent on the integrity of the reproductive system. Although several reproductive effects have been reported, but the degree of causality established between the abnormalities observed and exposure to particular chemicals is variable, and understanding of the mechanism(s) is limited. Fishes are a vital source of proteins and lipids for humans and domestic animals, forming the basis for economically important fisheries and aquaculture. Large efforts have recently been denoted to dissect the mechanisms of action of xenobiotics in aquatic species, with the ultimate aim of detecting, controlling and possibly intervening in chemical exposure and its effects on the aquatic ecosystem and humans. In this context, we ought to be concerned with the health and safety of aquatic species per se, as well as a resource for human needs.

Changes in three hepatic cytochrome P450 subfamilies during a reproductive cycle in turbot (Scophthalmus maximus L.)

Sexual differentiation of the hepatic cytochrome P450 system was characterized in 2-year-old farmed turbot (Scophthalmus maximus L.) during their first spawning period (January–September). The fish were kept in tanks supplied with continuously flowing seawater (34.5%, ppt) at a constant temperature of 16°C and natural photoperiod (60°N). Sampling of liver samples (n = 4–6) was performed once every month for 9 months. Pronounced sex differences were recorded in the activities of 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), and NADPH-cytochrome P450 reductase during spawning (May–July). EROD activity in female fish decreased gradually towards the onset of ovulation in May–July to rise again in the postspawning period. The decrease correlated with increasing gonadosomatic index and estradiol17β (E2) levels in plasma. Immunochemical detection of CYP1A (58 kDa), CYP2K-like (47 and 52 kDa), and CYP3A-like (58 and 60 kDa) proteins in Western blotting, and ELISA showed higher protein levels in male compared to female fish from April/May-June, and significant differences were observed in June (CYP2K-like also in April and May). Analysis of monthly variations within sexes during the reproductive cycle shows significant monthly changes in all parameters in both female and male fish. Both CYP2K- and CYP3A-like protein levels were significantly elevated in male fish during spawning in June. To study the induction response during spawning, β-naphthoflavone (BNF) 75 mg/kg body weight) was administered intraperitoneally to both sexes in June. BNF caused a significant increase in EROD and ECOD activities and CYP1A protein levels but had no effect on NADPH-cytochrome P450 reductase activity or CYP2K-like/CYP3A-like proteins. This study documents, for the first time in any fish species or lower vertebrate, the sexual differentiation in the liver of three different CYP subfamilies during sexual maturation and spawning. J. Exp. Zool. 277:313–325, 1997.

Plasma levels of vitellogenin and eggshell zona radiata proteins in 4-nonylphenol and o,p′-DDT treated juvenile Atlantic salmon (Salmo salar)

Abstract Induction of vitellogenin (Vtg) in males and juveniles of oviparous vertebrates has been used as a biomarker for xenoestrogens. Recently, we have demonstrated that synthesis of eggshell zona radiata proteins (Zrp) or zonagenesis is an integral aspect of fish oogenesis (Oppen-Berntsen et al. (1994) Journal of Experimental Zoology 268, 59–70), and that Zrp synthesis is a sensitive biomarker for nonylphenol (Arukwe et al. (1997) Environmental Health Perspective 105, 418–422). This study compares the responses of Vtg and Zrp in plasma of juvenile Atlantic salmon (Salmo salar) treated with 4-nonylphenol (NP) and o,p′-DDT (both at 25 mg kg−1, singly and in combination). Validated ELISA and immunoblot analysis show that Vtg and Zrp respond significantly stronger to NP treatment alone and in combination with o,p′-DDT compared to control and o,p′-DDT treatment alone. However, a slight reduction in NP-induced Zrp levels was indicated when NP was injected in combination with o,p′-DDT.

Fish model for assessing the in vivo estrogenic potency of the mycotoxin zearalenone and its metabolites.

The in vivo estrogenic potency of zearalenone (ZEA), a mycotoxin produced by different strains of Fusarium fungi, and its metabolites (alpha- and beta-zearalenol), have been studied in fish. Estrogenicity was evaluated using an in vitro competitive receptor binding assay and in vivo induction of vitellogenesis and zonagenesis, two estrogen receptor (ER)-mediated responses that are integral aspects of fish oogenesis. The ER binding affinities of alpha-zearalenol and ZEA in rainbow trout (Oncorhynchus mykiss) were approximately 1/150 and 1/300 to that of estradiol, respectively. Juvenile salmon (Salmo salar) were exposed to a single intraperitoneal injection of ZEA, alpha-zearalenol and beta-zearalenol (each at 1 and 10 mg/kg) and compared to fish injected with estradiol-17 beta (E2; 5 mg/kg) and controls. Using indirect enzyme-linked immunosorbent assay (ELISA) with homologous antibodies, a dose-dependent induction of vitellogenin (Vtg) and eggshell zona radiata proteins (Zr-proteins) were observed 7 days after exposure to ZEA and alpha-zearalenol. beta-Zearalenol did not elevate plasma Vtg levels, but a non-significant elevation of plasma Zr-proteins levels was observed at the highest dose (10 mg/kg). Generally, alpha-zearalenol and ZEA possess estrogenic potencies that are approximately 50% compared to that of E2, and their order of estrogenic potency (in both in vitro receptor competitive binding and in vivo induction of Vtg and Zr-proteins levels) is: alpha-zearalenol > ZEA > beta-zearalenol. Our results show that blood plasma analysis of Vtg and Zr-proteins levels provides a suitable in vivo fish model for assessing the estrogenic potencies of ZEA and its metabolites.

The expression of CYP1A, vitellogenin and zona radiata proteins in Atlantic salmon (Salmo salar) after oral dosing with two commercial PBDE flame retardant mixtures: absence of short-term responses

The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and cta-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days I and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n = 5 for each group) and 14 (n = 6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYPIA were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17beta for Vtg and Zrp expression, and beta-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.

Monoclonal and polyclonal antibodies against fish vitellogenin for use in pollution monitoring

Abstract Two monoclonal antibodies (KB-1 and BN-5) and one polyclonal antibody (AA-1) were prepared against purified vitellogenin (Vtg) from Atlantic salmon (Salmo salar), and tested for their ability to bind Vtg from plasma of various other fish species. The MAb KB-1 was found to bind specifically to Vtg from the Salmo species, Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). The MAb BN-5 showed a much wider cross-reactivity, binding to Vtg from both Salmoniformes species (Atlantic salmon, brown trout, rainbow trout and Arctic charr) and Pleuronectiformes species (turbot and halibut). The widest crossreactivity was observed with the polyclonal antibody AA-1 which, in addition to the species recognized by the MAb BN-5, also bound to Vtg from Atlantic cod (Gadiformes).

Salmon Eggshell Protein Expression: A Marker for Environmental Estrogens

Abstract: A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17β (E2) was constructed and screened with antibodies raised against salmon eggshell (zona radiata) proteins. Two clones, SalZr2_19 and SalZr2_23 were sequenced and shown to encode proteins of approximately 50 kDa. SalZr2_23 contains 12 octamer sequence lpqr/kpa/vq repeats also found in SalZr2_19, but only twice. Alignment reveals that the two salmon sequences are similar to piscine zona radiata proteins and mammalian zona pellucida proteins. Several transcripts ranging from 2.3 to 12 kb appeared in liver extracts of E2-treated fish. Juvenile fish treated with E2 or 4-nonylphenol showed strong induction of zona radiata protein. The use of egg envelope transcriptional and translational induction in male or juvenile fish as a biological marker of environmental estrogens is discussed.