Effect of seawater transfer on CYP1A gene expression in rainbow trout gills.

During the transfer of rainbow trout from freshwater to seawater, the gills have to switch from an ion-absorption epithelium to an ion-secretion epithelium in order to maintain equilibrium of their hydromineral balance. After a change to ambient salinity, several gill modifications have already been demonstrated, including ion transporters. In order to identify new branchial mechanisms implicated in seawater acclimation, we carried out an extensive analysis of gene expression in gills using microarray technology. This strategy allowed us to show that CYP1A gene expression was up-regulated in the gills after salinity transfer. This increase was confirmed by real-time reverse transcription PCR. Furthermore, measurements of CYP1A enzyme activity (EROD) showed a significant increase after transfer to seawater. Immunohistochemistry analysis in the gills revealed that cells with a higher expression of CYP1A protein were principally pillar cells and those in the primary lamellae not in contact with the external medium. The results of this study suggest for the first time that CYP1A may be implicated in the seawater acclimation of the gills of rainbow trout.

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 microg 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 microg l(-1) bNF. On the other hand, the induction of oestrogen-dependent proteins with 0.1 microg l(-1) EE2 was inhibited by exposure to 4.0 microg 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.

Variability of physicochemical and biological parameters between replicated outdoor freshwater lentic mesocosms.

Micro- and mesocosms are frequently required in regulatory procedures of aquatic risk assessment for pesticides. However, many questions are still a matter of debate with regard to the use of these systems for environmental risk assessment, especially considering the inter-system variability of the measured parameters and its consequences on experimental design and data analysis. In this paper, variability of physico-chemical and biological parameters measured during two long-term experiments (8 to 9 months) in uncontaminated outdoor freshwater lentic mesocosms (8 m3) is analysed. Consequences on the design of ecotoxicity tests in mesocosms and on data analysis are also addressed. Water temperature, pH, dissolved oxygen concentration and concentration of suspended solids exhibited a very low variability whereas nutrient concentrations displayed elevated levels of variability. Among biological parameters, those measured at the individual level were less variable than those measured at the community level. Functional descriptors frequently exhibited a lower inter-mesocosm variability than structural descriptors. Aggregation of data proved to significantly reduce inter-mesocosm variability. The results indicate that univariate statistical methods may be used for physico-chemical or species-level (e.g. biometric parameters) data which exhibit a moderate inter-mesocosm variability. The use of multivariate techniques is suggested for other levels of investigation. Nevertheless, variability is not sufficient to identify useful parameters. The sensitivity towards chemicals and ecological relevance of descriptors within the experimental context must also be considered.

Alkaline comet assay in rainbow trout hepatocytes.

The alkaline comet assay was performed to measure DNA integrity in fish hepatocytes. Primary cultures of rainbow trout hepatocytes were exposed to two known genotoxic compounds, hydrogen peroxide (H(2)O(2)) and benzo[a]pyrene (B[a]P), and to organic extracts of river sediments. The DNA damage in the form of single-strand breaks was monitored following the formation of DNA comets after alkaline electrophoresis. Exposure of the hepatocytes to H(2)O(2) for 2 hr increased strand breaks in a dose-related manner at the concentration range reported previously in studies with mammalian hepatocytes. B[a]P treatment led to a significant increase in strand breaks at the concentrations ranging from 0.1 to 10 muM after 4 hr of exposure. After 48 hr of exposure to B[a]P, the level of DNA strand breaks was lower than that of the control. The organic extracts obtained from river sediments significantly increased DNA strand breaks in trout hepatocytes, indicating the presence of genotoxic compounds in the sediment. The results show that the alkaline comet assay is a promising method by which to study the genotoxic potential of xenobiotics found in the aquatic environment.

The I protein of the heterogeneous nuclear ribonucleoprotein complex is a novel dog nuclear autoantigen.

In eukaryotic cells, heterogeneous nuclear RNA is associated with a set of abundant nuclear proteins to form complex ribonucleoprotein structures (hnRNP). Autoantibodies to hnRNP G protein have been previously reported in German shepherd dogs with lupus-like syndrome. In the present study, we describe the characterization of a novel antigen recognized by a serum from a schnauzer dog with a non-erosive polyarthritis. The autoantibodies give, by indirect immunofluorescence, a nuclear pattern with staining close to one of the nucleoli. Immunoblotting and immunoprecipitation data reveal that the autoantigens are in fact two closely related basic proteins (average pI 8.7) with apparent molecular weights of 56 kDa (p56) and 59 kDa (p59). The results of immunoprecipitation with anti-hnRNP antibodies and DNA affinity column chromatography strongly suggest that these autoantigens correspond to hnRNP I proteins. This point was confirmed by cloning and sequencing a cDNA clone encoding the complete sequence of the antigens. In addition, we found that anti-hnRNP I antibodies preferentially stain certain loops of the Pleurodeles waltl lampbruch chromosomes. These data, added to previous ones on anti-p43/hnRNP G protein in German shepherd dogs with lupus-like syndrome, confirm the interest of this category of antibodies to hnRNP proteins in autoimmune disorders.

Biotransformation enzyme activities in the olfactory organ of rainbow trout (Oncorhynchus mykiss). Immunocytochemical localization of cytochrome P4501A1 and its induction by ß-naphthoflavone.

Olfaction is a crucial function in most fish species, but little is known about biotransformation enzymes in the olfactory organ. This study demonstrates that biotransformation enzymes usually found in the rainbow trout liver, are present in the olfactory organ as well. While microsomal cytochrome P450 reductase, p-nitrophenol hydroxylase and cytosolic glutathioneS-transferase presented similar levels in both the olfactory organ and the liver, microsomal 7-ethoxyresorufinO-deethylase (EROD), 7-ethoxycoumarinO-deethylase, and 7-pentoxyresorufinO-deethylase were much lower in the olfactory organ (77-, 35-, 200-times respectively). Furthermore, microsomes from the olfactory organ were able to perform testosterone hydroxylation only in the 16α-position while testosterone was hydroxylated in the 16ß-position by liver microsomes. Using polyclonal antibodies raised against perch cytochrome P4501A1, the immunoreactive protein was shown to be strongly expressed in various cellular types forming the nonsensory epithelium. Some immunostaining was also reported in the nonsensory cellular elements constituting the sensory epithelium, while olfactory receptor cells failed to show cytochrome P4501A1-immunoreactivity. Finally, the exposure of rainbow trout to waterborne ß-naphthoflavone (0.1 µg ml(-1)) for 2 or 4 days resulted in a higher induction of EROD activity in the olfactory organ compared to the liver. The presence of biotransformation enzymes in the olfactory organ of rainbow trout addresses the question of their involvement in the detoxication/toxication of pollutants as well as in the olfactory function.

hnRNP G: sequence and characterization of a glycosylated RNA-binding protein.

The autoantigen p43 is a nuclear protein initially identified with autoantibodies from dogs with a lupus-like syndrome. Here we show that p43 is an RNA-binding protein, and identify it as hnRNP G, a previously described component of heterogeneous nuclear ribonucleoprotein complexes. We demonstrate that p43/hnRNP G is glycosylated, and identify the modification as O-linked N-acetylglucosamine. A full-length cDNA clone for hnRNP G has been isolated and sequenced, and the predicted amino acid sequence for hnRNP G shows that it contains one RNP-consensus RNA binding domain (RBD) at the amino terminus and a carboxyl domain rich in serines, arginines and glycines. The RBD of human hnRNP G shows striking similarities with the RBDs of several plant RNA-binding proteins.

DNA adduct detection: some applications in monitoring exposure to environmental genotoxic chemicals.

In the assessment of genotoxic risk factors in the environment, the measurement of DNA adducts in aquatic organisms and in plants may have considerable implications. Using 32P-postlabelling, we have detected DNA adducts in the liver of carp (Chondrostoma nasus) from the River Rhône (France), both downstream and upstream from a polychlorinated biphenyl incineration plant. Some of the DNA adducts were specific to downstream fish, suggesting a differential pattern of exposure. We have also detected DNA damage in needles in a declining spruce forest. We found that, in the declining forest, the amounts of DNA adducts increase in relation to the degree of damage to the needles whereas, in a healthy forest, the levels of DNA adducts were low. We have also found DNA adducts in the leaves of hops grown in fields where heptachlor residues persisted.

Nuclear distribution of PCNA during embryonic development in Xenopus laevis: a reinvestigation of early cell cycles.

The immunocytological distribution of the proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication, has been examined during the early development of Xenopus laevis. The protein is uniformly detected in nuclei during early stages up to the neurula stage. PCNA is detected by its distinctive cyclical pattern during early development, remaining detectable only during the period of S phase of each cell cycle. Immunological detection of PCNA is therefore a useful and specific non-isotopic marker of S-phase cells in the embryo. PCNA associates with typical karyomeric structures, suggesting that DNA replication starts before the nuclear compartment is entirely formed. At the midblastula transition, a new pattern of PCNA staining becomes apparent. First, a new type of PCNA staining is detected at the nuclear periphery. Second, mitotic clusters with different PCNA distributions suggest that the onset of desynchronization of the cell cycle at this stage is not random.

Neuropsychological symptoms and occupational exposure to anaesthetics.

OBJECTIVE: To analyse the relation between symptoms regularly reported by hospital personnel and exposure to anaesthetics. SETTING: Personnel of 18 hospitals in Paris from 1987 to 1989. DESIGN: An exposed group that included all operating theatre members except for doctors, and which was divided into three subgroups depending on the degree of exposure--exposure was measured by the frequency of the use of the scavenging system--and a control group that included other hospital personnel matched by hospital, sex, occupation, age, and duration of service. SUBJECTS: 557 exposed workers and 566 unexposed workers. MAIN OUTCOME MEASURES: The groups were compared according to the crude rates of regular symptoms. Adjusted odds ratios were calculated to estimate the risks associated with exposure to anaesthetic gas. Liver transaminase activities (alanine aminotransferase, aspartate aminotransferase (s-ASAT, and gamma-glutamyl transpeptidase) were measured and compared between groups of exposure. RESULTS: After controlling for working conditions and matching factors, neuropsychological symptoms and tiredness were reported more by workers in less often scavenged theatres than by controls. No difference was found between workers of the well scavenged theatres and controls. Among the exposed workers, the members of paediatric surgical staffs reported a higher rate of neurological complaints (tingling, numbness, cramps) and tiredness than the members of the other surgical staffs. They had a high value of s-ASAT more frequently than the other exposed workers. CONCLUSION: These results strengthen the hypothesis of a causal relation between exposure to anaesthetics and neuropsychological symptoms, and show a dose-response effect. They suggest that the use of ventilating systems in operating rooms is an effective means of prevention.

Glucocorticoid-mediated potentiation of P450 induction in primary culture of rainbow trout hepatocytes.

Induction of 7-ethoxyresorufin O-deethylase activity (a cytochrome P450IA-dependent activity) by beta-naphthoflavone (0.36 microM) is increased 2-3-fold by dexamethasone or cortisol (10(-9)-10(-7) M) in rainbow trout hepatocyte cultures. This potentiation does not seem to be a time-dependent process and could be a classical glucocorticoid receptor-mediated event resulting in enhanced transcriptional activation of the CYP1A, as previously shown in mammals. Since glucocorticoid levels can increase in fish exposed to pollutants, such steroids may interfere with the induction response to xenobiotics.

DNA adduct formation and 7-ethoxyresorufin O-deethylase induction in primary culture of rainbow trout hepatocytes exposed to benzo[a]pyrene.

The formation of DNA adducts, using the (32)P-postlabelling assay, and induction of 7-ethoxyresorufin O-deethylase (EROD) were investigated in a primary culture of rainbow trout hepatocytes exposed to benzo[a]pyrene (B[a]P). Concentrations of 0.1 and 1 mum-B[a]P were shown to induce EROD whereas 10 mum was an inhibitory concentration. DNA adducts were detected for 12 hr to 72 hr after exposure to 1 mum-B[a]P whereas EROD activity was increased 36 hr after treatment. The pattern of adducts was shown to be identical to that obtained after B[a]P treatment of rainbow trout in vivo, as demonstrated by co-chromatography of the adducts. Pre-exposure of hepatocytes for 48 hr to beta-naphthoflavone (betaNF) and subsequent 24-hr exposure to 1 mum-B[a]P did not lead to increased DNA adduct formation although betaNF treatment led to a 3.4-fold induction of EROD activity at the time of B[a]P addition. This study suggests that primary culture of rainbow trout hepatocytes provides a suitable method for studying DNA adduct formation and its modulating factors in vitro.

Use of the fish cytochrome P-450-dependent 7-ethylresorufin O-deethylase activity as a biochemical indicator of water pollution. Study of the liver and the kidney of male and female nase (Chondrostoma nasus) from the River Rhône.

7-Ethylresorufin O-deethylase (EROD) activity and the cytochrome P-450 content of liver and kidney microsomes were measured in male and female nase (Chondrostoma nasus) from the River Rhône (France) caught downstream and upstream of a PCB incineration plant. Concurrently, PCB concentrations in the flesh of nase were also measured. The results showed that sex affected hepatic, but not renal, EROD activity during gonadal maturation. The male nase demonstrated higher activity than the female. Upstream/downstream comparisons clearly revealed a more elevated hepatic EROD activity in fish contaminated by PCB pollution than in fish captured in the reference area throughout the reproductive cycle, demonstrating the reliability of this enzymatic activity as a biochemical indicator. Renal EROD activity did not seem to be sensitive to PCB pollution, since neither male nor female nase showed significant upstream/downstream differences.

Effect of beta-naphthoflavone and MCPA on liver and kidney drug-metabolizing enzymes from the carp, Cyprinus carpio.

The effects of beta-naphthoflavone (beta-NF) and a chlorophenoxyacetic acid herbicide (MCPA) on hepatic and renal monooxygenase activities and conjugating enzymes from immature carp (Cyprinus carpio) were studied. beta-NF increased hepatic monooxygenase activities but the patterns of differential induction generally obtained in rat liver microsomes with two series of homologous substrates, alkoxycoumarins and alkylresorufins, were not found to be similar in carp liver microsomes. On the other hand, MCPA caused no changes in oxidative metabolism, with the exception of decreased aryl hydrocarbon hydroxylase activity. Renal activities were not modified by MCPA, while beta-NF treatment resulted in marked increases in monooxygenase activities with alkylresorufins as substrates. No changes were found in conjugation activities after treatment with MCPA or beta-NF. These results indicate that (a) the herbicide MCPA should have no effect on drug-metabolizing enzymes from carp, and (b) the hepatic and renal monoxygenase activities of carp are responsive to beta-NF, allowing their use in monitoring water pollution.

Effects of chemical pollution on the activities of hepatic xenobiotic metabolizing enzymes in fish from the River Rhône.

Polychlorobiphenyl (PCBs) levels and hepatic xenobiotic metabolizing enzyme activities were measured in fish from three locations of the River Rhône to study the consequences of a constant loading of PCBs from a PCB incineration plant. Our results show that levels of PCBs and enzyme activities were higher in fish living downstream from the plant than in fish from two locations upstream, suggesting enzyme induction by PCBs (known to be potent inducers in laboratory conditions). Enzyme activities were studied in spring and autumn in three species: nase (Chondrostoma nasus), roach (Rutilus rutilus) and grayling (Thymallus thymallus). Induction was observed for three cytochrome P-450-dependent monooxygenase activities (MO), i.e. 7-ethoxyresorufin O-deethylase (EROD), aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD). There was a close correlation between EROD and AHH activities (for all species). Glutathione S-transferase activities were also shown to be related to the PCB levels. Conversely, cytochrome P-450 content and benzphetamine N-demethylase activity were not "PCB level-dependent". This study clearly demonstrates a close relationship between PCB contamination and MO activities in fish from the field and thus clearly emphasizes the interest in MO as a monitoring tool for estimating water quality.

Effect of gamma-hexachlorocyclohexane (lindane) on carp (Cyprinus carpio). I. Effect of chronic intoxication on humoral immunity in relation to tissue pollutant levels.

Lindane, the gamma-isomer of hexachlorocyclohexane, one of the most widely used insecticides, was incorporated into carp food for 109 days. The effects of 10, 100, or 1000 ppm of lindane in food on hematocrits and on antibody production against Yersinia ruckeri bacterin in sera and mucus were followed for 79 days. No effect of lindane on these parameters and on spleen weight was observed. Lymphoid organs, spleen and kidney, were as highly contaminated at the beginning of the inoculation (Day 30) as at the end of the experiment. The tissue levels of lindane in liver, spleen, kidney, or whole body, determinated after 30 and 109 days of contamination, indicated that the tissue distribution was dependent on organ lipid contents.

Effect of gamma-hexachlorocyclohexane (lindane) on carp (Cyprinus carpio). II. Effects of chronic intoxication on blood, liver enzymes, and muscle plasmic membrane.

A 30-day ingestion of gamma-hexachlorocyclohexane (lindane) by carp (Cyprinus carpio) induced hypoglycemia without activation of two hepatic gluconeogenesis enzymes (fructose diphosphatase, EC 4.1.2.13, and glucose-6-phosphatase, EC 3.1.3.9) and hyponatremia and variations in muscle plasmic membrane-bound enzymes (especially cholinesterases, EC 3.1.1.7). After 109 days carps exhibited a decrease in natremia but no significant hypoglycemia. There was an activation of gluconeogenesis enzymes. Important changes were observed in the activities of muscle plasmic membrane enzymes (especially 5'-nucleotidase, EC 3.1.3.5, and ATPases, EC 3.6.1.3). Lindane, a lipophilic substance, especially disturbed the activity of membrane-bound enzymes enclosed in a phospholipid matrix.

Characterization of some monooxygenase activities and solubilization of hepatic cytochrome P-450 in two species of freshwater fish, the nase (Chondrostoma nasus) and the roach (Rutilus rutilus).

1. Hepatic monooxygenase activities were studied in microsomal fractions from two species of freshwater fish, the nase (Chondrostoma nasus) and the European roach (Rutilus rutilus). 2. These activities were determined by using four substrates, 7-ethoxycoumarin, 7-ethoxyresorufin, benzo(a)pyrene, and 2,5-diphenyloxazole and were characterized according to pH, temperature, cofactors, and the differential effects of two inhibitors, metyrapone and alpha-naphthoflavone. 3. Solubilization of microsomes was achieved by the use of detergents, with a good recovery of the cytochrome P-450.

PCB and p,p'-DDE in Lake Geneva brown trout (Salmo trutta L.), and their use as bioenergetic indicators.

Gross growth efficiency of Lake Geneva brown trout (Salmo trutta) was estimated by monitoring age-related PCB and p,p'-DDE accumulation, food contamination and annual growth.Estimation was carried out using 3-, 4-, and 5-yr old fish. Roach (Rutilus rutilus) were determined as the main component of trout diet. Estimated growth efficiency coefficients were 0.4 (3-4 yr old fish) and 0.17 (4-5 yr old fish). Nevertheless numerous parameters can affect the result; their respective influences are discussed.Related to fish age, PCB and p,p'-DDE concentrations increase if expressed on a wet weight basis, but not if expressed on a lipid weight basis.At the same age, organochlorine residue concentrations keep similar values for trout and roach in spite of different trophic levels. Growth must be considered as a more dilution factor of PCB and p,p'-DDE in trout compared with roach.