The cytochrome P4501C (CYP1C) gene subfamily was recently discovered in fish, and zebrafish (Danio rerio) CYP1C1 transcript has been cloned. Here we cloned the paralogous CYP1C2, showing that the amino acid sequence is 78% identical to CYP1C1, and examined gene structure and expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2. Xenobiotic response elements were observed upstream of the coding regions in all four genes. Zebrafish adults and embryos were exposed (24 hours) to 100 nM 3,3',4,4',5-polychlorinated biphenyl (PCB126) or 20 ppm acetone and subsequently held in clean water for 24 hours (adults) or 48 hours (embryos). All adult organs examined (eye, gill, heart, liver, kidney, brain, gut, and gonads) and embryos showed basal expression of the four genes. CYP1A was most strongly expressed in liver, whereas CYP1B1, CYP1C1, and CYP1C2 were most strongly expressed in heart and eye. CYP1B1 and the CYP1C genes showed an expression pattern similar to one another and to mammalian CYP1B1. In embryos CYP1C1 and CYP1C2 tended to have a higher basal expression than CYP1A and CYP1B1. PCB126 induced CYP1A in all organs, and CYP1B1 and CYP1C1 in all organs except gonads, or gonads and brain, respectively. CYP1C2 induction was significant only in the liver. However, in embryos all four genes were induced strongly by PCB126. The results are consistent with CYP1C1 and CYP1C2, as well as CYP1A and CYP1B1, being regulated by the aryl hydrocarbon receptor. While CYP1A may have a protective role against AHR agonists in liver and gut, CYP1B1, CYP1C1, and CYP1C2 may also play endogenous roles in eye and heart and possibly other organs, as well as during development.

Four pulp and paper mills discharge their effluents in the same section of the Biobio River in central southern Chile. Pulp mill effluents are a very complex mixture with characteristics that depend on the type of raw material, the process technology, and the effluent treatment. To investigate the effect of pulp mill effluent discharges, immature Oncorhynchus mykiss were exposed to river sediments in the laboratory for 29 d. Three sampling areas were defined in a spatial gradient in the river: Preimpact, impact, and postimpact zones relative to the pulp and paper mill discharge areas. Ethoxyresorufin-O-deethylase activities were significantly higher in fish exposed to impact and postimpact sediments when compared to those exposed to preimpact sediments, and higher levels of vitellogenin were observed in the plasma of female fish exposed to impact and postimpact sediments. Histological analysis of the gonadal tissue showed an induction of gonadal maturation in fish exposed to sediment coming from the impact and postimpact zones (oocytes in a vitellogenic state). No site differences were observed in erythrocytes, although differences were noted in the leukocytes in the exposure areas. Finally, the biomarker approach showed evidence that the sediment associated with pulp mill effluent discharges produces some effects in fish under laboratory conditions.

Endocrine disruption (ED) effects due to pulp and paper mill effluents extracts involving different industrial procedures and effluent treatments (nontreated, primary, and secondary treated) were evaluated using immature triploid rainbow trout in a pulse-exposure toxicity experiment. The protocol involved the use of intraperitoneal injection of mill extracts (solid-phase extraction [SPE]) corrected for individual fish weight and included several laboratory standards (steroidal hormones and phytosterols). Biological endpoints at two different levels of biological organization were analyzed (molecular and individual organism). Results indicated that nonsignificant changes were observed in the individual physiological indices represented by condition factor, liver somatic index, and gonad somatic index during the experiment. Significant induction of liver ethoxyresorufin-O-deethylase activity was observed between different effluent treatments and experimental controls. Significant endocrine-disrupting effects at the reproductive level were observed in all effluent treatments involving significant increments in plasma vitellogenin (VTG) levels. Fish exposed to untreated effluent extracts had significantly higher VTG levels compared to fish exposed to primary and secondary treatment effluent extracts, indicating a decrease of the estrogenic effect due to the effluent treatment. The present study has shown that for the Chilean pulp and paper mill SPE extracts evaluated, an endocrine disruption effect was induced in immature triploid rainbow, reaffirming the significant estrogenic effects demonstrated previously in laboratory and field experiments.

Caging experiments were conducted using hatchery-reared, immature, female rainbow trout (Oncorhynchus mykiss) in three previously defined areas of the Biobio River (south central Chile) representing a pollution gradient from the pulp and paper mill discharges area: a pre-impact area (upstream area, reference location), an impact area (area directly influenced), and a postimpact area (downstream area, less influenced). No significant changes were observed in the physiological index as represented by condition factor (K) and liver somatic index during different sampling times (after 11, 21, and 30 d of exposure). Ethoxyresorufin-O-deethylase activities were significantly higher in trout caged at the impact and postimpact discharges areas (two- to fourfold) compared with the reference (pre-impact) area, and a strong inhibition of acetylcholinesterase activity, reaching 50%, was observed mainly in fish caged at the impact area. A significant endocrine-disrupting effect (reproductive level) was evidenced by significant increments in gonad somatic index and plasma vitellogenin levels combined with an induction of gonad maturation (presence of vitellogenic oocytes) in trout caged at the impact and postimpact areas. These results, generated by an in situ approach, confirmed our group's findings for trout exposed to sediment in the laboratory: discharges of pulp mill effluent in the Biobio River are associated with the effects evaluated at different biological levels.

This study investigates factors affecting reproduction in fish exposed to pulp and paper mill effluents by comparing effluents from countries with varying levels of documented effects. To explore the hypothesis of wood as a common source of endocrine disrupting compounds, feedstocks from each country were analyzed. Analyses included in vitro assays for androgenic activity (binding to goldfish testis androgen receptors), estrogenic activity (yeast estrogen screen), and neurotransmitter enzyme inhibition (monoamine oxidase and glutamic acid decarboxylase). Chemical analyses included conventional extractives, known androgens, and gas chromatograph index (GCI) profiles. All effluents and wood contained androgenic activity, particularly in nonpolar fractions, although known androgens were undetected. Effluents with low suspended solids, having undergone conventional biotreatment had lower androgenic activities. Estrogenic activity was only associated with Brazilian effluents and undetected in wood. All effluents and wood inhibited neurotransmitter enzymes, predominantly in polar fractions. Kraft elemental chlorine free mills were associated with the greatest neurotransmitter inhibition. Effluent and wood GCI profiles were correlated with androgenic activity and neurotransmitter enzyme inhibition. Differences in feedstock bioactivities were not reflected in effluents, implying mill factors mitigate bioactive wood components. No differences in bioactivities could be discerned on the basis of country of origin, thus we predict effluents in regions lacking monitoring would affect fish reproduction and therefore recommend implementing such programs.

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Patterns of fish community composition in a south-central Chile river were investigated along the altitudinal-spatial and environmental gradient and as a function of anthropogenic factors. The spatial pattern of fish communities in different biocoenotic zones of the Chillan River is influenced by both natural factors such a hydrologic features, habitat, and feeding types, and also by water quality variables which can reduce the diversity and abundance of sensitive species. A principal component analysis incorporating both water quality parameters and biomarker responses of representative fish species was used to evaluate the status of fish communities along the spatial gradient of the stream. The abundance and diversity of the fish community changed from a low in the upper reaches where the low pollution-tolerant species such as salmonid dominated, to a reduced diversity in the lower reaches of the river where tolerant browser species such as cypriniformes dominated. Even though the spatial pattern of fish community structure is similar to that found for the Chilean Rivers, the structure of these communities is highly influenced by human disturbance, particularly along the lower reaches of the river.