A gill filament-based assay for the cytochrome P450 1A (CYP1A)-catalysed activity ethoxyresorufin O-deethylase (EROD) was developed in rainbow trout (Oncorhynchus mykiss) and applied to Atlantic salmon (Salmo salar), Arctic charr (Salvelinus alpinus), Atlantic cod (Gadus morhua), saithe (Pollachius virens), and spotted wolffish (Anarhichas minor). Exposure to waterborne β-naphthoflavone (βNF; 10-6 M) induced branchial EROD activity in all species but the spotted wolffish. In rainbow trout exposed to low concentrations of benzo[a]pyrene (BaP; 10-9 M) and the textile dye indigo (10-8 M) the gills responded more rapidly than the liver to BaP, and indigo induced branchial but not hepatic EROD activity. A CYP1A-dependent BaP adduct formation was shown in gills of fish exposed to waterborne 3H-BaP, i.e. the adduct formation was enhanced by βNF and blocked by ellipticine (CYP1A inhibitor). The predominant location for BaP adducts was the secondary lamellae (most exposed part of the gill filament), whereas the CYP1A enzyme was also present in the primary lamellae of the gill filament. Hence, in addition to the cell-specific expression of CYP1A an important determinant for the localisation of adducts seemed to be the bioavailability of BaP. This idea is supported by the fact that the CYP1A enzyme was induced only in secondary lamellae by BaP (10-7 M) and indigo (10-6 M), whereas it was induced in both primary and secondary lamellae by 3,3´,4,4´,5-pentachlorobiphenyl (10-8 M). Apparently, readily metabolised inducers (BaP and indigo) are biotransformed in the secondary lamellae. My results show that gill filament EROD activity is a sensitive biomarker of exposure to waterborne dioxin-like pollutants, and that the assay has potential for use in monitoring. Furthermore, the results suggest that readily metabolised dioxin-like compounds absorbed via the gills may undergo first-pass metabolism in the gill cells and therefore remain undetected by monitoring of EROD activity in the liver.

Induction of the cytochrome P450(CYP)1A protein and the connected increase in 7-ethoxyresorufin O-deethylase (EROD) activity are common biomarkers in fish. Enhanced activity of this protein signals exposure to Ah-receptor agonists such as chlorinated dioxins, co-planar polychlorinated biphenyls (PCBs) and certain polycyclic aromatic hydrocarbons (PAHs). The EROD biomarker is commonly analyzed in liver microsomes. However, the gill is directly exposed to waterborne pollutants, and in this thesis the gill filament EROD assay was therefore evaluated as a monitoring tool for waterborne CYP1A inducers in fish. Originally developed in rainbow trout (Oncorhynchus mykiss), the assay was here applied in various limnic and marine species. Following exposure to low waterborne concentrations of the readily metabolized CYP1A inducers benzo(a)pyrene (BaP) and indigo, a strong EROD induction was observed in the gill but not in the liver. This likely reflected metabolic clearance of the inducers in gill and other extrahepatic tissues. The high sensitivity of the gill was confirmed in studies of fish caged in waters in urban and rural areas in Sweden where the gill consistently showed a more pronounced EROD induction compared with the liver and the kidney. Fish caged in the reference waters showed surprisingly strong gill EROD induction and CYP1A immunostaining. Consequently, there may be CYP1A inducers present in the aquatic environment that are not yet identified. The assay was further applied in Atlantic cod (Gadus morhua) as a biomarker of exposure to crude oil and produced water (PW) from oil fields in the North Sea. The assay was finally adapted to detect inhibiting compounds, and an imidazole, a triazole and a plant flavonoid turned out to be potent gill EROD inhibitors. The overall conclusion from the studies of this thesis is that the gill filament EROD assay is a practical and sensitive biomarker of exposure to waterborne CYP1A inducers in various fish species. The induction of gill EROD activity in fish also at the reference sites in the field studies calls for further studies on background contamination in Swedish waters.

Exposure to crude oils has been shown to induce CYP1A enzymes and cause chronically toxic effects in aquatic organisms. Earlier studies indicated that polycyclic aromatic hydrocarbons (PAHs) present in crude oil are primarily responsible for the chronic toxicity. Crude oil contains a variety of PAHs; the majority of them are alkyl substituted. In this work, we have used an effects-driven fractionation and analysis approach (EDFA) to isolate and characterize PAHs present in Alaskan North Slope and Scotian Light crude oils that are toxic to fish. The crude oil components were first fractionated into four fractions using a low temperature vacuum distillation technique. Among them, the heavy gas oil fraction (boiling range 287°C – 461°C) of both oils caused highest toxicity to fish. To isolate the PAHs from waxes present in this fraction, a low temperature wax precipitation method was developed and optimized. CYP1A induction results showed that the extract contained a large number of CYP1A inducers while the residue contained none. Chemical analyses confirmed that most of the PAHs were partitioned into the extract fraction. Alkyl PAHs present in the extract were further fractionated into five fractions based on the number of aromatic rings using a normal phase HPLC method. Chemical analysis and the toxicity testing of these fractions indicated that alkyl PAHs belonging to classes such as phenanthrene, fluorene, naphthobenzothiophene, and chrysene are likely responsible for the observed toxic effects. To aid the EDFA scheme, a new HPLC-DAD method for the analysis of alkyl PAHs was developed. The alkyl PAHs were first fractionated based on the number of aromatic rings using a normal phase column followed by their analysis using reverse phase HPLC–DAD technique. The reverse phase analysis involved classifying the alkyl PAH peaks into different PAH classes based on their DAD spectra. Then, alkyl carbon numbers for each peak were assigned based on their retention time. To analyze co-eluting alkyl PAH isomers an offline multi-dimensional HPLC method was developed. Orthogonal separation was achieved by first fractionating the alkyl PAHs on a normal phase column followed by the RP-HPLC-DAD analysis. Using these data a 2D contour plot was developed and used for the detailed analysis of alkyl PAHs isomers. Analysis results showed good agreement with a gas-chromatography-mass-spectrometric (GC-MS) analysis method, and the new method was able to distinguish some PAH types which could not be identified by GC-MS. / Thesis (Ph.D, Chemistry) -- Queen's University, 2007-11-19 13:46:18.602

Numerous contaminants are present in mixtures in the aquatic environment. Among these are the azoles, a group of chemicals that includes both pharmaceuticals and pesticides. Azole fungicides are designed to inhibit lanosterol 14-demethylase (cytochrome P450 (CYP) 51), while other azoles are intended to inhibit aromatase (CYP19), i.e. the enzyme catalyzing biosynthesis of estrogens. In fish, a variety of CYP enzymes are involved in biotransformation of waterborne contaminants, and in metabolism of endogenous compounds including steroidal hormones. The induction of CYP1A protein and 7-ethoxyresorufin O-deethylase (EROD) activity are common biomarkers for exposure to aryl hydrocarbon receptor (AhR) agonists in fish. We developed an assay to measure inhibition of CYP1A activity (EROD) in three-spined stickleback and rainbow trout gill tissue ex vivo. Several azole fungicides were found to be potent inhibitors of CYP1A activity. A wastewater effluent containing high concentrations of pharmaceuticals was also shown to inhibit CYP1A activity. Further, several azoles inhibited CYP19 activity in rainbow trout brain microsomes in vitro. Azole mixtures reduced both CYP1A and CYP19 activity monotonically and in an additive way. Given the additive action of the azoles, studies to determine adverse effects of azole mixtures on CYP-regulated physiological functions in fish are needed. Induction of EROD and of gene expression of CYP1 in several organs was observed in an in vivo exposure with the same effluent shown to inhibit EROD. This finding could imply that there was a mixture of AhR agonists and CYP1A inhibitors in the effluent. Finally, wastewater treatment technologies were evaluated using biomarker responses in rainbow trout exposed to effluents of different treatments. The results from chemical analysis together with the biomarker results show that ozone and granulated active carbon treatment removed most pharmaceuticals, as well as AhR agonists and other chemicals present in the regular effluent. This part of the thesis demonstrates that biomarkers in fish such as induction of CYP1 gene expression are applicable to evaluate the efficiency of different treatment technologies for wastewater.

Submitted by dayse paz (daysepaz@hotmail.com) on 2016-04-08T01:42:53Z No. of bitstreams: 1 Disserta_o_C_ntia_da_Cruz_Chivittz.pdf: 1143457 bytes, checksum: fb5e304bb41926719c86894c487a6544 (MD5) / Approved for entry into archive by cleuza maria medina dos santos (cleuzamai@yahoo.com.br) on 2016-04-13T22:45:53Z (GMT) No. of bitstreams: 1 Disserta_o_C_ntia_da_Cruz_Chivittz.pdf: 1143457 bytes, checksum: fb5e304bb41926719c86894c487a6544 (MD5) / Made available in DSpace on 2016-04-13T22:45:54Z (GMT). No. of bitstreams: 1 Disserta_o_C_ntia_da_Cruz_Chivittz.pdf: 1143457 bytes, checksum: fb5e304bb41926719c86894c487a6544 (MD5) Previous issue date: 2014 / A poluição dos ambientes aquáticos por contaminantes orgânicos e misturas complexas, como aquelas provenientes de efluentes domésticos e industriais, tem levado certas populações de peixes a apresentarem adaptações bioquímicas e moleculares. Dentre estas, estão o aumento nos níveis do citocromo P450 1A (CYP1A), e em algumas populações, a perda da sua capacidade de indução após exposição a contaminantes agonistas do receptor de hidrocarbonetos aromáticos (AHR). Tal impossibilidade de induzir CYP1A é denominada resposta refratária, e vem sendo estudada há decadas em certas populações de peixes nativos da América do Norte. O presente estudo avaliou as respostas transcricionais de CYP1A utilizando RT-qPCR em brânquia, fígado e gonopódio dos peixes guarús Sul-Americanos Jenynsia multidentata e Phalloceros caudimaculatus coletados no entorno do estuário da Lagoa dos Patos, RS, Brasil. Os níveis de CYP1A em J. multidentata e P. caudimaculatus de um local contaminado por efluentes domésticos e outro próximo ao polo petroquímico, respectivamente, foram mais altos do que em locais referência, distantes destas fontes de contaminação. Exemplares de J. multidentata, provenientes de quatro locais foram expostos a 1 µM de β-naftoflavona (BNF) durante 24 h; e obtiveram indução de CYP1A em todos os órgãos em relação aos controles, exceto no fígado de peixes de um local contaminado, o que indica a possível existência de resposta refratária de CYP1A. Os resultados sugerem que os mecanismos responsáveis pela adaptação de J. multidentata aos locais poluídos são de alguma forma semelhantes aos encontrados em peixes da América do Norte e, possivelmente, envolve a resposta refratária de CYP1A e polimorfismos do AHR. O presente estudo forneceu informações a respeito da adaptação envolvendo respostas refratárias de CYP1A em peixes de locais poluídos e dá suporte para a utilização dos níveis transcricionais de CYP1A em guarús Sul-Americanos como biomarcador para o monitoramento da contaminação ambiental. / The water pollution caused by organic contaminants and complex mixtures, such as domestic and industrial sewage, caused some fish species to adapt at the biochemical and molecular level. This adaptation includes the increase in the level of cytochrome P450 1A (CYP1A) and an absence of induction by compounds that are agonists of the aryl hydrocarbon receptor (AHR). This impossibility to induce CYP1A is called refractory response of the CYP1A, and has been studied by decades in some native fish populations in North America. The present study evaluated the transcriptional responses of CYP1A using RT-qPCR in gills, liver and gonopodium of the South American guppies Jenynsia multidentata and Phalloceros caudimaculatus collected around the Patos Lagoon Estuary, RS, Brazil. The level of CYP1A in J. multidentata and P. caudimaculatus collected in a stream contaminated with domestic sewage discharges and from an area close to a petrochemical center, respectively, showed high levels of CYP1A expression when compared to reference locations distant from the these sources of contamination. Copies of J. multidentata, from four sites were exposed to 1 µM of β-naftoflavona (BNF) for 24 h; and obtained induction of CYP1A in all organs compared to controls, except for the liver of fish from one contaminated site, which indicates the possible existence of CYP1A refractory response. The results suggest that the mechanisms responsible for the adaptation of J. multidentata to live in polluted sites are somehow similar to those found in North American fish and possibly are also related to the CYP1A refractory response and AHR receptor polymorphisms. This study provided informations about the adaptation involving refractory responses of CYP1A in fish from polluted environments and give support for the use of CYP1A transcriptional levels in South American guppies as biomarkers for the monitoring of environment contamination.

Made available in DSpace on 2016-06-02T19:29:26Z (GMT). No. of bitstreams: 1 3129.pdf: 1211425 bytes, checksum: fe662cd2f2192794e3ea63b146284d10 (MD5) Previous issue date: 2010-05-19 / Universidade Federal de Sao Carlos / The aim of this work was to evaluate the effects of the water-soluble fraction of gasolina (WSFG) to the Neotropical fish Prochilodus lineatus. The WSFG was prepared by adding gasoline to water (1:4) this mixture was then exposed to intense sunlight for 6h, simulating a gasoline spill in tropical conditions. After that the upper insoluble phase was discharged and the WSFG was collected. Fish were exposed for 6, 24 and 96h to the WSFG diluted to 5% (EXP group) or only to water (control group or CTR). The following parameters were analyzed: biochemical (antioxidants and EROD) of gills and liver, hematologic, osmo-ionic, metabolic, endocrine (cortisol) besides the density and distribution of chloride cells (CC) and the activity of gills Na+/K+-ATPase (NKA). The increased in ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase (GST) activity indicated phase I and II biotransformation of the compounds present in the WSFG in both organs. The activation of CYP1A in the gills pointed out the importance of this organ in the biotransformation of xenobiotics. The liver showed an increase in reduced glutathione (GSH) content at 24 and 96 h exposure to WSFG and the increase in the activity of catalase (CAT) and glutathione peroxidase (GPx) after 96 h exposure. The gills showed an activation of the antioxidant defenses with an increased CAT activity soon after 6h exposure and an increase in GSH content after 24h exposure. However, for both organs the antioxidants defense was not enough to prevent oxidative damage, as shown by the occurrence of lipid peroxidation in liver and gills after 6 and 96h of exposure, respectively. The WSFG also promoted hemolysis, as indicated by the changes in the hematological parameters analyzed and an increase in plasma K+. Fish showed a secondary stress response, noted by the occurrence of hyperglycemia in all the periods of exposure, despite no significant increase in plasma cortisol. The WSFG also lead to an increase in the density of CC, in the activity of NKA, in plasma concentrations of Na+ and in the osmolarity in fish exposed to WSFG for 24h. Taken together these results showed that the compounds present in the WSFG interfere on the functioning of vital organs such as liver and gills of Prochilodus lineatus. / O objetivo deste trabalho foi avaliar os possíveis efeitos da fração solúvel da gasolina (FSG) em alguns parâmetros bioquímicos e fisiológicos do peixe neotropical Prochilodus lineatus. A FSG foi preparada misturando-se gasolina em água (1:4), essa mistura foi exposta à radiação solar intensa durante 6 h, simulando um derrame de gasolina em condições tropicais. Após, a FSG foi coletada e a fração insolúvel foi descartada. Os animais foram expostos por 6, 24 e 96 h à FSG diluída 5 % (grupo EXP) ou apenas à água (grupo controle ou CTR). Foram analisados parâmetros bioquímicos (antioxidantes e indução da CYP1A) em brânquia e fígado, hematológicos, osmo-iônicos, metabólicos, endócrino (cortisol) além da densidade e distribuição de células-cloreto (CC) e a atividade da enzima Na+/K+-ATPase (NKA) nas brânquias. O aumento na atividade da etoxiresorufina-O-desetilase (EROD) e da glutationa-S-transferase (GST) indicou a estimulação das vias de biotransformação de fase I e II dos compostos da FSG em ambos os órgãos. A ativação das enzimas de detoxificação nas brânquias ressaltou a importância deste órgão na biotransformação de xenobióticos. O fígado apresentou aumento na concentração de glutationa reduzida (GSH) após 24 h e 96 h de exposição à FSG e aumento na atividade catalase (CAT) e glutationa peroxidase (GPx) após 96 h de exposição. As brânquias mostraram uma ativação das vias antioxidantes com o aumento da CAT logo após 6 h de exposição, e da concentração de GSH após 24 h de exposição. No entanto, para ambos os órgãos estudados, a ativação das defesas antioxidantes não foi suficiente para impedir os danos oxidativo, como indicado pela ocorrência de peroxidação lipídica (LPO) no fígado e nas brânquias após 6 e 96 h de exposição, respectivamente. A FSG também provocou hemólise comprovada pela diminuição dos parâmetros hematológicos analisados, seguido pelo aumento do K+ plasmático. Os peixes mostraram uma resposta secundária de estresse visualizado pela ocorrência da hiperglicemia em todos os períodos de exposição, apesar da ausência de diferenças significativas na concentração plasmática do cortisol. A FSG também provocou aumento na densidade das CC, na atividade da NKA, nas concentrações plasmáticas do Na+ e osmolaridade nos animais expostos durante 24 h. Esses resultados em conjunto indicam que os compostos presentes na FSG afetaram de maneira significativa órgãos vitais como o fígado e as brânquias do Prochilodus lineatus.

Many chemicals present in the aquatic environment can interfere with physiological functions in fish. Exposure to chemicals can be revealed by the use of biomarkers. Induction of 7-ethoxyresorufin O-deethylase (EROD) activity is a commonly used biomarker for exposure to CYP1A inducers such as dioxins and polyaromatic hyrdrocarbons. Vitellogenin is a frequently used biomarker for estrogenic compounds in various fish species whereas a biomarker for androgens, spiggin, is only found in sticklebacks. The main objectives of this thesis were to evaluate gill EROD activity as a biomarker and the three-spined stickleback as a model species in ecotoxicological studies. EROD activities were measured in gill, liver and kidney in rainbow trout (Oncorhynchus mykiss) caged in urban areas in Sweden. EROD induction was most pronounced in the gill. Also in fish caged at reference sites, with an expected low level of known CYP1A inducers, a marked gill EROD induction was found. One suggested inducer in rural waters is humic substances (HS). To evaluate the EROD-inducing capacity of HS, three-spined sticklebacks (Gasterosteus aculeatus) were exposed to HS of natural or synthetic origin. Both kinds of HS caused significant EROD induction. Gill EROD activities were also induced in sticklebacks exposed to ethynylestradiol (EE2) and β-naphthoflavone (βNF), alone and in combinations. Production of vitellogenin was induced in sticklebacks exposed to ≥50 ng EE2/l and a significant decrease in spiggin production was observed in individuals exposed to 170 ng EE2/l. Results from this thesis further strengthen the contention that gill EROD activity is a very sensitive biomarker for CYP1A inducers and that the stickleback is a suitable biomonitoring species, especially for exposure to CYP1A inducers. The finding that not only classical CYP1A inducers but also HS and high EE2 concentrations stimulate gill EROD activity is of significance for the interpretation of biomonitoring data.

The effects of diabetes on cytochrome P450 dependent drug metabolizing enzymes have not to be clarified yet. The most widely used animals in these studies have been rats, and information regarding the effects of diabetes on cytochrome P450 dependent procarcinogen/carcinogen metabolism in rabbits is limited. In the present study, we investigated, for the first time, the influence of benzene on liver, kidney and lung microsomal cytochrome P450 dependent drug metabolizing enzyme activities, protein and mRNA levels in diabetic and non-diabetic rabbits. Male New Zealand rabbits were made diabetic by a single dose of alloxan treatment in this study. AST, ALT and LDH enzyme activities in the blood serum and lipid peroxidation in liver microsomes were found to increase in diabetic, benzene treated and benzene treated diabetic rabbits. Besides these, CYP2E1 dependent NDMA N-demethylase and p-nitrophenol hydroxylase activities and CYP2E1 protein level were found to increase in liver and kidney of diabetic and benzene-treated rabbits. The combined effects of benzene and diabetes on these activities and protein level were found to be additive. Although diabetes caused induction of pulmonary CYP2E1 protein level and associated enzyme activities, benzene treatment of rabbits resulted in no change in enzyme activities and protein level in lung. The level of mRNA was investigated by Real-Time PCR. Accordingly, hepatic CYP2E1 mRNA level was increased 6.71-, 10.53- and 12.93-fold in diabetic, benzene treated and benzene treated diabetic rabbits with respect to the control animals. Similarly, renal CYP2E1 mRNA level was found in increase in these rabbits. In addition to CYP2E1, CYP3A6 associated enzyme activity, erythromycin N-demethylase, CYP3A6 protein and mRNA level were found to increase in diabetic rabbit liver and lung. Unlike diabetes, benzene treatment caused suppression of CYP3A6 protein and inhibition of associated enzyme activity in liver. There was no significant change in the erythromycin N-demethylase activity and CYP3A6 level of liver and lung as a result of benzene treatment of diabetic rabbits. Moreover, diabetes induced CYP1A2 protein and mRNA level and CYP1A associated enzyme activities in the rabbit liver. On the other hand, benzene caused statistically insignificant decreases in CYP1A dependent enzyme activities and CYP1A2 protein level in liver. CYP1A associated enzyme activities, CYP1A2 protein and mRNA levels were not changed in the liver of benzene treated diabetics. The results of the present work indicate that both diabetes and benzene stimulate metabolic activation toxic chemicals metabolized by CYP2E1 such as NDMA and benzene by inducing CYP2E1 which results in the formation of increased amounts of reactive metabolites. Application of benzene to diabetic rabbits further elevates expression and activities of the CYP2E1. As a result of additive induction of the CYP2E1 in benzene treated diabetics, further increase the risk of hepatotoxicity produced by toxins may be observed when compared to the separate treatments. This may in turn further potentiate the risk of organ toxicity and mutagenesis in liver and kidney of these subjects. As in the case of CYP2E1, the risk of carcinogenesis due to induction of CYP1A may be increased in diabetic subjects. Moreover, in diabetic and benzene exposed subjects, alteration of drug clearance and clinical drug toxicity may be observed due to induction or suppression of CYP3A.

<p>The polychlorinated biphenyls (PCBs) comprise a group of 209 congeners varying in the number of chlorine atoms and substitution patterns. These compounds tend to be biomagnified in foodwebs and have been shown to induce an array of effects in exposed organisms. The structural characteristics of the PCBs influence their potency as well as mechanism of action. In order to assess the biological potency of these compounds a multi-step quantitative structure-activity relationship (QSAR) procedure was used in the project described in this thesis.</p><p>The ultraviolet absorption (UV) spectra were measured for all 209 PCBs, and digitised for use as physico-chemical descriptors. Interpretations of the spectra using principal component analysis (PCA) showed the number of ortho chlorine atoms and para-para substitution patterns to be significant. Additional physico-chemical descriptors were derived from semi-empirical calculations. These included various molecular energies, the ionisation potential, electron affinity, dipole moments, and the internal barrier of rotation. The internal barrier of rotation was especially useful for describing the conformation of the PCBs on a continuous scale.</p><p>In total 52 physico-chemical descriptors were compiled and analysed by PCA for the tetra- to hepta-chlorinated congeners. The structural variation within these compounds was condensed into four principal properties derived from a PCA for use as design variables in a statistical design to select congeners representative for these homologue-groups. The 20 selected PCBs have been applied to study structure-specific biochemical responses in a number of bioassays, and to study the biomagnification of the PCBs in various fish species.</p><p>QSARs were established using partial least squares projections to latent structures (PLS) for the PCBs potency to inhibit intercellular communication, activate respiratory burst, inhibit dopamine uptake in synaptic vesicles, compete with estradiol for binding to estrogen receptors, and induce cytochrome P4501A (CYP1A) related activities. By the systematic use of the designed set of PCBs the biological potency was screened over the chemical domain of the class of compounds. Further, sub-regions of highly potent PCBs were identified for each response measured. For risk assessment of the PCBs potency to induce dioxin-like activities the predicted induction potencies (PIPs) were calculated. In addition, two sets of PCBs were presented that specifically represent congeners of environmental relevance in combination with predicted potency to induce estrogenic and CYP1A related activities.</p>

The polychlorinated biphenyls (PCBs) comprise a group of 209 congeners varying in the number of chlorine atoms and substitution patterns. These compounds tend to be biomagnified in foodwebs and have been shown to induce an array of effects in exposed organisms. The structural characteristics of the PCBs influence their potency as well as mechanism of action. In order to assess the biological potency of these compounds a multi-step quantitative structure-activity relationship (QSAR) procedure was used in the project described in this thesis. The ultraviolet absorption (UV) spectra were measured for all 209 PCBs, and digitised for use as physico-chemical descriptors. Interpretations of the spectra using principal component analysis (PCA) showed the number of ortho chlorine atoms and para-para substitution patterns to be significant. Additional physico-chemical descriptors were derived from semi-empirical calculations. These included various molecular energies, the ionisation potential, electron affinity, dipole moments, and the internal barrier of rotation. The internal barrier of rotation was especially useful for describing the conformation of the PCBs on a continuous scale. In total 52 physico-chemical descriptors were compiled and analysed by PCA for the tetra- to hepta-chlorinated congeners. The structural variation within these compounds was condensed into four principal properties derived from a PCA for use as design variables in a statistical design to select congeners representative for these homologue-groups. The 20 selected PCBs have been applied to study structure-specific biochemical responses in a number of bioassays, and to study the biomagnification of the PCBs in various fish species. QSARs were established using partial least squares projections to latent structures (PLS) for the PCBs potency to inhibit intercellular communication, activate respiratory burst, inhibit dopamine uptake in synaptic vesicles, compete with estradiol for binding to estrogen receptors, and induce cytochrome P4501A (CYP1A) related activities. By the systematic use of the designed set of PCBs the biological potency was screened over the chemical domain of the class of compounds. Further, sub-regions of highly potent PCBs were identified for each response measured. For risk assessment of the PCBs potency to induce dioxin-like activities the predicted induction potencies (PIPs) were calculated. In addition, two sets of PCBs were presented that specifically represent congeners of environmental relevance in combination with predicted potency to induce estrogenic and CYP1A related activities.