Biomedical subjects

A Arukwe

Publications and source records attributed to A Arukwe.

13 recordsLinked to original sources

PubMed2002

Regio-specific hydroxylation of nonylphenol and the involvement of CYP2K- and CYP2M-like iso-enzymes in Atlantic salmon (Salmo salar).

Previously, it has been demonstrated that nonylphenol (NP) has a dual function in regulating reproductive hormones by: (1) increasing the activity of steroid metabolizing enzymes at low concentration, that does not induce vitellogenin (Vtg) and zona radiata proteins and (2) decreasing the activity of these enzymes at higher concentration [Environ. Health Perspect. 105 (1997) 109; Environ. Toxicol Chem. 16 (1997) 2576]. To more precisely understand the estrogenic effects of NP in fish and a possible interference with steroid hormone metabolism, we investigated in the Atlantic salmon the identity of the cytochrome P450 enzymes involved in NP hydroxylation. Up to 9 metabolites were separated by radio-HPLC when [R-(14)C]-4n-NP was incubated with hepatic microsomes from juvenile salmon. In control fish the major metabolites were identified as monohydroxylated products at omega-, (omega-1)- and (omega-2)-positions of the alkyl chain of 4n-NP. Salmon hepatic microsomes formed omega-, (omega-1)- and (omega-2)-lauric acid (LA) hydroxylation products. The potency of alpha-naphthoflavone, ketoconazole and ethynylestradiol (non-specific, but strong inhibitors of CYP1A, 2K and 3A, respectively) on LA and NP hydroxylations were assessed in the present study. Ketoconazole inhibited omega-, (omega-1)- and (omega-2)-hydroxylations of LA and 4n-NP and was the only inhibitor of omega-hydroxylation of both substrates. Ethynylestradiol specifically inhibited (omega-1)- and (omega-2)-hydroxylations of LA as well as 4n-NP. Interestingly, the lowest NP dose (1 mg/kg) was the most potent inducer of NP-metabolites formation. These results suggest the involvement of CYP2M- and 2K-like enzymes in terminal and subterminal hydroxylations of 4n-NP respectively, and was confirmed by the competitive inhibition between LA and 4n-NP. The production of one unidentified 4n-NP metabolite was not affected by any of the chemicals used, suggesting a possible ring hydroxylation with involvement of another cytochrome P450 isoform. Our data reveal a novel aspect of CYP isozymes involvement in NP metabolism that may complicate the assessment of its endocrine effects. Hence, the regio-selective hydroxylation of endocrine disruptors, such as NP, by CYP isozymes is revealed as a possible new marker of estrogenicity.

Animals↗

PubMed2001

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.

Androgens↗

PubMed2001

In vivo modulation of nonylphenol-induced zonagenesis and vitellogenesis by the antiestrogen, 3,3'4,4'-tetrachlorobiphenyl (PCB-77) in juvenile fish.

Zonagenesis and vitellogenesis (eggshell zona radiata protein (Zrp) and vitellogenin (Vtg) production, respectively), are two estrogen-regulated processes in oviparous vertebrates that are crucial for oocyte maturation. Treatment of juvenile Atlantic salmon (Salmo salar) with nonylphenol (NP; 25 mg kg(-1)) alone or in combination with 3,3',4,4'-tetrachlorobiphenyl (TCB; 0.1 mg kg(-1)) resulted in pronounced elevations of plasma eggshell Zrp and Vtg and their respective liver mRNA levels in two separate experiments. TCB treatment alone caused the elevation of CYP1A mRNA, protein and enzyme levels (7-ethoxyresorufin O-deethylase (EROD)). In experiment 3, which also included the time factor, exposure of juvenile salmon to 10 and 25 mg NP per kg in combination with TCB generally resulted in reduced plasma Zrp and Vtg levels, compared with NP treatments alone. In a fourth experiment, juvenile salmon were exposed to different doses of TCB either 2 days before or 2 days after a single dose (25 mg kg(-1)) of NP. Samples were always collected 5 days after the NP exposure and analyzed for mRNA and protein levels. Generally, TCB doses given 2 days after NP exposure resulted in the elevation of Vtg and Zrp protein and mRNA levels. Vtg and Zrp mRNA levels were also elevated in the groups treated with 0.1 mg TCB 2 days before NP exposure. In all experiments, TCB injection resulted in the induction of liver CYP1A mRNA, CYP1A protein and EROD activity, but no Zrp or Vtg protein/mRNA inductions were observed when given alone. The present study documents for the first time the apparent stimulation of xenoestrogen-induced responses by an antiestrogenic CYP1A-inducer, in fish or any other lower vertebrate. However, the stimulatory or inhibitory effect of TCB on NP-induced responses appear to be dependent on the ratio of NP and TCB doses, and temporal sequence of exposure. Fish hepatic zonagenesis and vitellogenesis continue to provide interesting models for further studies on the mechanisms and possible interactions between endocrine disruptors and CYP1A-inducers, their antiestrogenic and/or estrogen potentiating effects.

Journal Article↗

PubMed2001

Differential biomarker gene and protein expressions in nonylphenol and estradiol-17beta 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.

Animals↗

PubMed2000

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.

Journal Article↗

PubMed2000

Effects of xenoestrogen treatment on zona radiata protein and vitellogenin expression in Atlantic salmon (Salmo salar).

Zonagenesis (zona radiata protein synthesis) and vitellogenesis (yolk protein synthesis) are two reproductive responses that are integral aspects of fish oogenesis. This study examines the responses of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) to five environmental pollutants; 4-nonylphenol (NP) and o,p'-DDT [both at 25 mg/kg], lindane (gamma-HCH) [10 mg/kg], a technical PCB mixture (Aroclor 1254; A1254) and bisphenol A (BPA) [both at 5 mg/kg] in juvenile Atlantic salmon (Salmo salar). Fish were given intraperitoneal injections of o,p'-DDT, gamma-HCH, A1254 or BPA; singly, in combination with NP, and as a cocktail of all five chemicals, and later compared to NP-treated and untreated fish. In a separate experiment, fish were exposed to BPA in a dose-response manner (1, 5, 25 and 125 mg/kg fish). Based on previous studies, blood and liver samples were collected 2 weeks after injection. Zrp and Vtg levels were analyzed in plasma using immunoblotting and enzyme-linked immunosorbent assay. Liver cytochrome P4501A was analyzed by 7-ethoxyresorufin O-deethylase (EROD) activity. NP caused pronounced elevations in plasma Zrp and Vtg levels. In comparison, when NP was given in combination with gamma-HCH, Vtg levels was significantly reduced, compared to NP treatment alone. Using o,p'-DDT, A1254 and BPA, significant elevations of plasma Zrp and Vtg were seen when chemicals were given in combination with NP, but not when administered by themselves. An apparent dose-response induction of Vtg and Zrp levels were observed in BPA treated juvenile salmon. In immunoblots, one component of molecular weight approximating the Zrp-beta was detected when either o,p'-DDT, gamma-HCH, A1254 or BPA were given singly. A1254 significantly induced hepatic EROD activity when administered alone. However, when given as a mixture with all the other xenobiotics, reduction of EROD activity was observed. The data suggest a pattern of xenobiotics action which may complicate assessment of their reproductive effects. Zrp (the beta monomer) was more responsive to the xenoestrogens than Vtg, and provides a sensitive means of detecting exposure to environmental estrogens.

Journal Article↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Salmon Eggshell Protein Expression: A Marker for Environmental Estrogens.

: A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17beta (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.

Journal Article↗

PubMed1997

Fish zona radiata (eggshell) protein: a sensitive biomarker for environmental estrogens.

Environmental estrogens have recently caused great concern because of their ability to mimic natural hormones and influence vital endocrine functions in humans and wildlife. The induction of vitellogenin (Vtg) synthesis by environmental estrogens in viviparous vertebrates has been proposed as an effective and sensitive biomarker of estrogenicity. Immunochemical analysis of plasma from Atlantic salmon (Salmo salar) exposed to 4-nonylphenol (NP) or to effluent from oil refinery treatment plant (ORTP), shows that NP and ORTP effluent induces Vtg and zona radiata proteins (Zrp) in a dose-dependent manner. However, Zrp-beta cross-reactive proteins are more responsive than Zrp-alpha, Zrp-gamma, and Vtg. The sensitivity of Zrp induction points to the zona radiata proteins as alternate biomarkers of estrogenicity.

Animals↗

PubMedDate not supplied

Metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar).

Nonylphenol (NP) is a breakdown product of alkylphenol polyethoxylates (APEs), an important class of non-ionic surfactants that are widely used in many detergent formulations and plastic products for industrial and domestic use. A complex microbial degradation pattern, characterized by the formation of several metabolic products that are more toxic than the parent compound, has been established for APEs. We have studied the in vivo metabolism and organ distribution of NP in juvenile salmon. Fish were exposed to a single oral dose of [3H]-4-n-NP (1295 KBq, 25 micrograms) and sampled at 24, 48 and 72 h after exposure. Metabolites were separated by radio-high-performance liquid chromatography and tentatively identified by cochromatography with standards characterized by mass spectrometry. Our results show that 4-n-NP was mainly metabolized in vivo to its corresponding glucuronide conjugate and to a lesser extent to various hydroxylated and oxidated compounds. Biliary excretion at 72 h after dosing amounted to 2.83 +/- 0.75% of the administered radioactivity. Kinetic analysis shows that NP-glucuronide accounted for 83, 95 and 81% of total radioactivity in the HPLC-injected bile sample at 24, 48 and 72 h, respectively, after exposure. The half-life of residues in carcass and muscle was between 24 and 48 h after exposure.

Animals↗

PubMedDate not supplied

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.

Administration, Oral↗

PubMedDate not supplied

Thyroid hormone-dependent gene expression as a biomarker of short-term 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) exposure in European common frog (Rana temporaria) tadpoles.

The effects on thyroid hormone-dependent gene biomarker responses of the persistent organochlorine pesticide metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) were investigated after exposure of 4-week-old European common frog (Rana temporaria) (stage 36) tadpoles to two (0.001 and 0.01 ppm) DDE concentrations. Total body weight, total length, and tail length and width increased after 3-day exposure to DDE. Expression patterns of genes encoding for growth hormone, thyroid-stimulating hormone (TSHbeta) and thyroid hormone receptor (TRalpha and TRbeta) isoforms were evaluated in the head, body and tail regions using a validated real-time polymerase chain reaction (PCR) method. The mRNA expression of growth hormone in the body, and TSHbeta in the head showed significant DDE concentration-dependent decreases. While DDE caused variable effects on TRalpha mRNA steady-state, the expression of TRbeta was significantly decreased in the tail by DDE in a concentration-specific manner. The effect of DDE exposure on TRbeta mRNA expression showed a negative correlation with tail length and width during the exposure period. The unique pattern of a DDE-induced decrease of tail TRbeta expression probably reflects the significant role of this thyroid hormone receptor isoform in tail re-absorption and overall metamorphosis in anuran species. Therefore, the present study shows that the evaluation of thyroid hormone-dependent genes may represent quantitative biomarkers of acute exposure to organochlorine pesticides in anuran species during critical developmental periods such as metamorphosis. Given the widespread environmental levels of DDT and its metabolites, these pollutants will remain a subject of concern and their effects on anuran species should be studied in more detail.

Animals↗