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This study developed a sensitive extraction method for urine matrix (based on lyophilization, without the need for pre-cleaning by solid phase extraction), coupled to LC-MS/MS analysis of the biomarker 8-hydroxy-2′ - deoxyguanosine (8-OHdG). The methodology was validated in urine samples from a cohort of Spanish pregnant women collected during the first, second and third trimester of pregnancy, and urine samples collected within 24 h after delivery (n = 85). A detection and quantification limit of 0.01 and 0.05 μg/L, respectively, were established. The median 8-OHdG concentration was 2.18 μg/L (range 0.33–7.79); and the corresponding creatinineadjusted concentrations ranged from 1.04 to 13.12 with median of 4.48 μg 8-OHdG/g creatinine. The concentrations of non-adjusted 8-OHdG significantly decreased (p < 0.05) in the 3rd trimester and post-delivery urine samples when compared to the 1st trimester levels. 8-OHdG concentrations were further studied in placenta samples matching the same urine samples (n = 26), with a median value of 1.3 ng 8-OHdG/g of tissue. Placental 8-OHdG concentrations were correlated with urinary levels of non-adjusted 8-OHdG in the 3rd trimester. Considering the small cohort size, results must be interpreted with caution, however statistical analyses revealed elevated urinary non-adjusted 8-OHdG levels in the 1st trimester of mothers that delivered boys compared to those who delivered girls (p < 0.01). Increased urinary non-adjusted 8-OHdG concentrations at the time of delivery were significantly associated with clinical records (any type of clinical record during pregnancy; p < 0.05). The novel extraction and analytical method for the assessment of 8-OHdG is applicable for sensitive analysis of multiple analytes or biomarkers in urine matrix. This method could also be applied for other matrices such as blood or tissues. Our findings show that 8-OHdG in urine of pregnant women could predict oxidative stress in placenta and can be related to characteristics such as maternal obesity, mode of delivery and newborn sex., European Union through Horizon 2020 projects HBM4EU [grant number 733032], CETOCOEN Excellence [grant number 857560], Horizon Europe project PARC [grant agreement No 101057014], RECETOX Research Infrastructure (No LM2023069), MEYS, and the OP RDE (the CETOCOEN EXCELLENCE project No. CZ.02.1.01/0.0/0.0/ 17_043/0009632)

Drinking water treatment ultimately aims to provide safe and harmless drinking water. Therefore, the suitability of a treatment process should not only be assessed based on reducing the concentration os a pollutant concentration but, more importantly, on reducing its toxicity. Hence, the main objective of this study was to answer whether the degradation of a highly toxic compound of global concern for drinking water equals its detoxification. We, therefore, investigated the treatment of cylindrospermopsin (CYN) by center dot OH and SO4-center dot produced in Fenton and Fenton-like reactions. Although SO4-center dot radicals removed the toxin more effectively, both radical species substantially degraded CYN. The underlying degradation mechanisms were similar for both radical species and involved hydroxylation, dehydrogenation, decarboxylation, sulfate group removal, ring cleavage, and further fragmentation. The hydroxymethyl uracil and tricyclic guanidine moieties were the primary targets. Furthermore, the residual toxicity, assessed by a 3-dimensional human in vitro liver model, was substantially reduced during the treatment by both radical species. Although the results indicated that some of the formed degradation products might still be toxic, the overall reduction of the toxicity together with the proposed degradation pathways allowed us to conclude: "Yes, degradation of CYN equals its detoxification!".

An electrochemical biosensor is presented that directly reflects the metabolic activity of prokaryotic and eukaryotic cells. This biosensor can be used measure the biological activity of bacteria, yeasts and mammalian cells. This makes the sensor interesting for various applications in industry and science. A possible application is bioprocess control, monitor activities from yeasts, bacteria and fungi to increase the yield. Other applications are starter culture quality studies in the food industry and cytoxicological evaluation with mammalian cells. Our latest investigations additionally indicate the applicability of the electrochemical biosensor to measure algae and cyanobacteria. In our investigations, we were able to show that it was also possible to detect photosynthetic organisms with the electrochemical measurement method, used for investigations on prokaryotic and eukaryotic organisms before. Therefore, this the present study demonstrates an alternative to using this electrochemical biosensor equipped with alga and cyanobacteria for toxicological investigations based on selected test chemicals. The results of this study show a good correlation with those from reference methods, such as the Algal Growth Inhibition Test and the Microtox Test. The advantages of the new electrochemical biosensor are easy handling and shorter measurement time by using different types of test organisms. The evaluation of the sensor signal is based on the current–time curves of a potentiostatic measurement produced by the detection of microbially reduced mediator molecules immobilized in a gel structure. The mediator molecules are reduced during the measurement process. The reduced mediator molecules produce a current signal, which rapidly provides information about the vigor and vitality of living bacteria, yeasts, fungi or cells.

Objectives The objective of this review paper was to stimulate collaborative discussions toward the development of a general concept of an open source protocol for a feasible and efficient longitudinal birth cohort study exploring non-communicable diseases (NCDs), their multifactorial etiology and relations between various risk factors. Study design The present paper systematically reviews the design of existing birth cohorts in Europe containing environmental exposure data, and assesses a quantity and quality of their research outcomes as their potential to be an effective tool for studying non-communicable diseases and their risk factors. Methods European birth cohorts with more than 3000 participants have been included in the study. A total number of scientific papers published in the internationally recognized journals and their impact factors and citation records were evaluated for all cohorts as surrogates for their efficiency to contribute to NCDs understanding and thus their prevention. Results The birth cohorts contributing most significantly to the NCD understanding shared common features: (i) study size between 10,000 and 15,000 mother–child pairs; (ii) repeated assessment of children from prenatal into adulthood; and (iii) availability of biological samples. Smaller cohorts and cohorts with a specific focus generated a lower number of publications; however, these often received considerably a higher number of citations. Conclusions General cohort studies with 10,000–15,000 mother–child pairs allow a broader context interpretation, publish a higher number of articles, and often lead to the formation of infrastructures for ‘spin-off (nested) studies’.

Rehabilitation of aged drinking water pipes is an extensive renovation and increasingly topical in many European cities. Spray-on-lining of drinking water pipes is an alternative cost-effective rehabilitation technology in which the insides of pipes are relined with organic polymer. A commonly used polymer is epoxy resin consisting of monomer bisphenol A (BPA). Leaching of BPA from epoxy lining to drinking water has been a concern among public and authorities. Currently epoxy lining is not recommended in some countries. BPA leaching has been demonstrated in laboratory studies but the behavior and ageing process of epoxy lining in situ is not well known. In this study 6 locations with different age epoxy linings of drinking water pipes done using two distinct technologies were studied. While bisphenol F, 4-n-nonylphenol, and 4-t-octylphenol were rarely found and in trace concentrations, BPA was detected in majority of samples. Pipes lined with the older technology (LSE) leached more BPA than those with more recent technology (DonPro): maxima in cold water were 0.25 μg/L and 10 ng/L, respectively. Incubation of water in pipes 8–10 h prior to sampling increased BPA concentration in cold water 1.1–43-fold. Hot water temperature caused even more BPA leaching - at maximum 23.5 μg/L. The influence of ageing of epoxy lining on BPA leaching on could be shown in case of LSE technology: locations with 8–9 years old lining leached 4–20-fold more BPA compared to a location with 2-year-old lining. Analysis of metals showed that epoxy lining can reduce especially iron concentration in water. No significant burden to water could be shown by the analyzed 72 volatile organic compounds, including epichlorhydrin, precursor used in epoxy resin. Estrogenicity was detected in water samples with the highest BPA loads. Comparable responses of two yeast bioreporters (estrogen receptor α and BPA-targeted) indicated that bisphenol-like compounds were the main cause of estrogenicity. Compared to the estimated average daily BPA exposure, additional BPA load via cold drinking water in the studied locations was low, maximum 8.7%. However, hot water should also be considered as exposure source due to higher BPA concentrations. Epoxy lined locations should be monitored in future in order to evaluate ageing process and control increasing leaching of potentially harmful chemicals.

Colonization of man-made surface materials by algae and other microorganisms, i.e. biofouling, causes various aesthetical and health problems as well as deterioration of appearance and surface functioning. The present study investigated eight complex surfaces combining various TiO2, alkoxy-siloxane and hydrophobization agents that were combined to prevent growth of phototrophic filamentous alga Klebsormidium sp. Regarding photoactivity, NOx abatement at the studied surfaces has been improved by adding anti-aggregation substances such as bentonite. Surface properties were characterized by confocal microscopy, scanning electron microscopy, SEM, with the energy dispersive X-ray spectroscopy, EDX, and the contact angle measurements. Biological experiments with Klebsormidium alga were performed in a constructed flow-through system. The results indicated that superhydrophobicity, i.e. very low surface energy, was critical for efficient antifouling during the early colonization by algae. This was specifically demonstrated in two novel composite samples made of titanium dioxide and commercial hydrophobization agent UltraEverDry. Low surface energy of these materials (around 1.3 mJ/m2), high water contact angles (>140°) as well as decent photoactivity (NOx conversions by 16.7 % and 1.4 % in UV and VIS, respectively), provide a promising basis for further longer-term anti-fouling experiments. The present study introduces novel composite materials preventing the algal fouling, and it brings new evidences on the interactive effects on hydrophobic and photoactive surfaces.

Herbicides and their metabolites are often detected in water bodies where they may cause adverse effects to non-target organisms. Their effects at environmentally relevant concentrations are often unclear, especially concerning mixtures of pesticides. This study thus investigated the impacts of one of the most used herbicides: S-metolachlor and its two metabolites, metolachlor oxanilic acid (MOA) and metolachlor ethanesulfonic acid (MESA) on the development of zebrafish embryos (Danio rerio). Embryos were exposed to the individual substances and their environmentally relevant mixture until 120 hpf (hours post-fertilization). The focus was set on sublethal endpoints such as malformations, hatching success, length of fish larvae, spontaneous movements, heart rate and locomotion. Moreover, expression levels of eight genes linked to the thyroid system disruption, oxidative stress defense, mitochondrial metabolism, regulation of cell cycle and retinoic acid (RA) signaling pathway were analyzed. Exposure to S-metolachlor (1 mu g/L) and the pesticide mixture (1 mu g/L of each substance) significantly reduced spontaneous tail movements of 21 hpf embryos. Few rare developmental malformations were observed, but only in larvae exposed to more than 100 mu g/L of individual substances (craniofacial deformation, non-inflated gas bladder, yolk sac malabsorption) and to 30 mu g/L of each substance in the pesticide mixture (spine deformation). No effect on hatching success, length of larvae, heart rate or larvae locomotion were found. Strong responses were detected at the molecular level including induction of p53 gene regulating the cell cycle (the pesticide mixture - 1 mu g/L of each substance; MESA 30 mu g/L; and MOA 100 mu g/L), as induction of cyp26a1 gene encoding cytochrome P450 (pesticide mixture - 1 mu g/L of each substance). Genes implicated in the thyroid system regulation (dio2, thra, thrb) were all overexpressed by the environmentally relevant concentrations of the pesticide mixture (1 mu g/L of each substance) and MESA metabolite (1 mu g/L). Zebrafish thyroid system disruption was revealed by the overexpressed genes, as well as by some related developmental malformations (mainly gas bladder and yolk sac abnormalities), and reduced spontaneous tail movements. Thus, the thyroid system disruption represents a likely hypothesis behind the effects caused by the low environmental concentrations of S-metolachlor, its two metabolites and their mixture.

Aerobic composting and anaerobic digestion plays an important role in reduction of organic waste by transforming the waste into humus, which is an excellent soil conditioner. However, applications of chemical-contaminated composts on soils may have unwanted consequences such as accumulation of persistent compounds and their transfer into food chains. The present study investigated burden of composts and digestates collected in 16 European countries (88 samples) by the compounds causing dioxin-like effects as determined by use of an in vitro transactivation assay to quantify total concentrations of aryl hydrocarbon receptor-(AhR) mediated potency. Measured concentrations of 2,3,7,8-Tetrachlorodibeno-p-dioxin (2,3,7,8-TCDD) equivalents (TEQbio) were compared to concentrations of polycyclic aromatic hydrocarbons (PAHs) and selected chlorinated compounds, including polychlorinated dibenzo-p-dioxins/furans (PCDD/ Fs), co-planar polychlorinated biphenyls (PCBs), indicator PCB congeners and organochlorine pesticides (OCPs). Median concentrations of TEQbio (dioxin-like compounds) determined by the in vitro assay in crude extracts of various types of composts ranged from 0.05 to 1.2 with a maximum 8.22 lg (TEQbio )k g 1 dry mass. Potencies were mostly associated with less persistent compounds such as PAHs because treatment with sulfuric acid removed bioactivity from most samples. The pan-European investigation of contamination by organic contaminants showed generally good quality of the composts, the majority of which were in compliance with conservative limits applied in some countries. Results demonstrate performance and added value of rapid, inexpensive, effect-based monitoring, and points out the need to derive corresponding effect-based trigger values for the risk assessment of complex contaminated matrices such as composts.

In vitro assays are broadly used tools to evaluate the estrogenic activity in Waste Water Treatment Plant (WWTP) effluents and their receiving rivers. Since potencies of individual estrogens to induce in vitro and in vivo responses can differ it is not possible to directly evaluate risks based on in vitro measures of estrogenic activity. Estrone, 17beta-estradiol, 17alfa-ethinylestradiol and to some extent, estriol have been shown to be responsible for the majority of in vitro estrogenic activity of municipal WWTP effluents. Therefore, in the present study safe concentrations of Estrogenic Equivalents (EEQs-SSE) in municipal WWTP effluents were derived based on simplified assumption that the steroid estrogens are responsible for all estrogenicity determined with particular in vitro assays. EEQs-SSEs were derived using the bioassay and testing protocol-specific in vitro potencies of steroid estrogens, in vivo predicted no effect concentration (PNECs) of these compounds, and their relative contributions to the overall estrogenicity detected in municipal WWTP effluents. EEQs-SSEs for 15 individual bioassays varied from 0.1 to 0.4 ng EEQ/L. The EEQs-SSEs are supposed to be increased by use of location-specific dilution factors of WWTP effluents entering receiving rivers. They are applicable to municipal wastewater and rivers close to their discharges, but not to industrial waste waters. Keywords: Estrogen, Threshold, In vitro assay, Environmental risk assessment, Waste water treatment plant

Despite intensive research into toxic bloom-forming cyanobacteria, the majority of their metabolites remain unknown. The present study explored in detail a novel bioactivity identified in cyanobacteria, i.e. inhibition of gap junctional intercellular communication (GJIC), a marker of tumor promotion. The extracellular mixture (exudate) of the cyanobacterial strain Cylindrospermopsis raciborskii (SAG 1.97) was fractionated by semi-preparative reversed phase HPLC, and the fractions assessed for their potencies to inhibit GJIC. Two non-polar fractions that significantly inhibited GJIC were further fractionated, tested and analyzed using multiple mass spectrometric methods. Investigations led to the identification of a putative chemical compound (molecular formula C18H34O3, m/z 299.2581 for the [M+H](+) ion) responsible for observed bioactivities. Specific inhibitors of signaling pathways were used to screen for biochemical mechanisms beyond GJIC inhibition, and the results indicate the involvement of ERK1/2 kinases via a mechanism related to the action of epidermal growth factor EGF but clearly distinct from other anthropogenic tumor promoters like polychlorinated biphenyls or polycyclic aromatic hydrocarbons. The chemical and in vitro toxicological characterizations of the newly described metabolite provide important insights into the still poorly understood health impacts of complex toxic cyanobacterial blooms and indicate that currently applied monitoring practices may underestimate actual risks.

Endocrine-disruptive potential and concentrations of polar organic contaminants were measured in seven headwaters flowing through relatively unpolluted areas of the Czech Republic. Towns with Wastewater Treatment Plant (WWTP) discharges were the first known sources of anthropogenic pollution in the areas. River water was sampled several kilometers upstream (US) and several tens of meters downstream (DS) of the WWTP discharges, by use of Pesticide and Pharmaceutical Polar Organic Integrative Samplers (POCIS-Pest, POCIS-Pharm). Extracts of passive samplers were tested by use of a battery of in vitro bioassays to determine overall non-specific cytotoxicity, endocrine-disruptive (ED) potential and dioxin-like toxicity. The extracts were also used for quantification of polar organics. There was little toxicity to cells caused by most extracts of POCIS. Estrogenicity was detected in all types of samples even though US locations are considered to be background. At US locations, concentrations of estrogen equivalents (EEq) ranged from less than the detection limits (LOD) to 0.5 ng EEq/POCIS. Downstream concentrations of EEqs ranged from less than LOD to 4.8 ng EEq/POCIS. Concentrations of EEq in POCIS extracts from all DS locations were 1 to 14 times greater than those at US locations. Concentrations of EEq measured in extracts of POCIS-Pest and POCIS-Pharm were in a good agreement. Neither antiestrogenic nor anti/androgenic activities were detected. Concentrations of 2,3,7,8-TCDD equivalents (TEqbio) were detected in both types of POCIS at concentrations ranging from less than the LOD to 0.39 ng TEqbio/POCIS. Nearly all extracts of POCIS-Pharm contained greater concentrations of TEqbio activity than extracts of POCIS-Pest. Concentrations of pesticides and pharmaceuticals in extracts of POCIS were generally small at all sampling sites, but levels of some pharmaceuticals were significantly greater in both types of POCIS from DS locations. Chemical analyses along with the results of bioassays documented impacts of small towns with WWTPs on headwaters. Keywords: Androgen, Dioxin-like activity, Estrogen, In vitro assay, POCIS, Waste Water Treatment Plant

Retinoids are dietary hormones acting through nuclear receptors for retinoic acid, important especially during embryonic development. This study focuses on the disruption of signaling pathways of retinoids by polycyclic aromatic hydrocarbons (PAHs) and their N-heterocyclic analogs (N-PAHs), important environmental contaminants with numerous biological effects. In vitro test with P19/A15 cell line stably transfected with luciferase reporter gene under control of retinoic acid-responsive elements was used to investigate both direct activation of retinoic acid receptors and modulation of response induced by natural ligand all-trans retinoic acid (ATRA) by 26 PAHs and N-PAHs. While none of individual compounds alone activated retinoic acid receptors, many of them modulated ATRA-mediated activity both after 6 h and 24 h exposure. Majority of compounds active after 6 h downregulated ATRA-mediated activity (most effective were two analogs of dibenz[a,h]anthracene with LOECs about 185 nM), while most compounds active after 24 h upregulated the effects of ATRA (most effective benz[a]acridine and dibenz[a,i]acridine caused 400% induction of ATRA response). Quantitative structure-activity relationship analysis identified molecular volume and dipole moment as the most important descriptors of inhibitory effects after 6 h, while length, total molecular energy, gap-HOMO/LUMO and Van der Waals energy are important descriptors for stimulatory effects of PAHs and N-PAHs. This study demonstrates those abundant pollutants such as PAHs and their analogs interfere in vitro with retinoid signaling, which could play role in some in vivo effects of these organic contaminants such as teratogenicity.

This work evaluated the effects of various cyanobacterial fractions in Frog Embryo Teratogenesis Assay Xenopus (FETAX) with African clawed frog embryos. Fractions were prepared from five biomasses with different dominant genera (Microcystis, Aphanizomenon, Anabaena, Planktothrix) and different microcystin content. Effects of following fractions were investigated: (I) homogenate of complex cyanobacterial biomass, (II) cell debris (pellet) after centrifugation of complex biomass, (III) supernatant after centrifugation of complex biomass (= crude aqueous extract), (IV) permeate after passing of crude extract through C-18 column (fraction devoid of microcystins), and (V) eluate from C-18 column (containing microcystins, if present). Besides classical parameters evaluated in 96 h FETAX (mortality, growth inhibition, malformations), we have also assessed the effects on biochemical markers of oxidative stress and detoxification (glutathione pool, GSH; activity of glutathione peroxidase, GPx; glutathione reductase, GR; activity of glutathione-S-transferase, GST). Complex biomass (I) and aqueous extract (III) were generally the most toxic fractions in terms of mortality and growth inhibition, whereas eluates containing microcystins (V) were generally less toxic. On the other hand, the same fraction (eluates) induced significant malformations in low concentrations but the effects were not related to the content of microcystins. Biomarkers were affected in variable manner but no significant effect or clear relation to microcystin content was observed. Our data support the hypothesis that microcystins are not the only or major toxic compounds in the complex cyanobacterial samples (at least for some species) and that more attention should be paid to other components of complex cyanobacterial biomass including non-specific parameters such as oxygen content or toxic ammonia released during bacterial decay of organic material.

Quaternary benzo[c]phenanthridine alkaloids (QBAs) sanguinarine and chelerythrine exert a plethora of biological activities. Nevertheless, the specific cellular target for these alkaloids within the cell was not identified as far. Several literary data indicate that biological effects of QBAs could be associated with aryl hydrocarbon receptor (AhR) signaling pathway, including cytochrome P450 CYP1A, however, available information are controversial. In this work we analyzed the effects of sanguinarine and chelerythrine on AhR activity in rat hepatoma cells HII4E. luc stably transfected with dioxin responsive element fused to luciferase gene (DRE-LUC). Studied QBAs were tested in submicromolar concentration range (0.0001–1 μM) and in incubation times 6, 24 and 48 h. Transcriptional activity of AhR was monitored by chemiluminiscence measurement of luciferase catalytic activity. Sanguinarine and chelerythrine did not activated AhR in any time or dose tested. Chelerythrine (1 μM) but not sanguinarine caused moderate inhibition of AhR activation by 10 picomolar dioxin (exponential phase of receptor activation). In contrast, AhR activation by 2.5 nM dioxin (saturated receptor) was not affected by either alkaloid tested. In conclusion, the findings presented here favor rather for inactivity or modest inhibitory effect of QBAs on AhR signaling pathways in vitro than for the activation of the receptor. Regarding the concentrations of QBAs occurring in vivo , the use of products containing sanguinarine and/or chelerythrine has low toxicological potential in terms of the interactions with AhR signaling pathways.

A group of intracellular nuclear receptors is a protein superfamily including arylhydrocarbon AhR, estrogen ER, androgen AR, thyroid TR and retinoid receptors RAR/RXR as well as molecules with unknown function known as orphan receptors. These proteins play an important role in a wide range of physiological as well as toxicological processes acting as transcription factors (ligand-dependent signalling macromolecules modulating expression of various genes in a positive or negative manner). A large number of environmental pollutants and other xenobiotics negatively affect signaling pathways, in which nuclear receptors are involved, and these modulations were related to important in vivo toxic effects such as immunosuppression, carcinogenesis, reproduction or developmental toxicity, and embryotoxicity. Presented review summarizes current knowledge on major nuclear receptors (AhR, ER, AR, RAR/RXR, TR) and their relationship to known in vivo toxic effects. Special attention is focused on priority organic environmental contaminants and experimental approaches for determination and studies of specific toxicity mechanisms.

Berberine has long been considered a candidate for an antimalarial drug. It exerts a plethora of biological activities and has been used in the treatment of diarrhea and gastro-enteritis for centuries. Here we provide evidence that berberine activates the aryl hydrocarbon receptor (AhR) in human hepatoma (HepG2) and rat hepatoma cells stably transfected with a dioxin responsive element fused to the luciferase gene (H4IIE.luc). AhR was activated by high doses of berberine (10-50 microM) after 6 and 24 h of incubation as revealed by CYP1A1 mRNA expression (HepG2) and AhR-dependent luciferase activity (H4IIE.luc). Berberine induced nuclear translocation of AhR-GFP chimera transiently transfected to Hepa1c1c7 cells. In contrast, low doses of berberine (

Activation of aryl hydrocarbon receptor (AhR) by 30 polycyclic aromatic hydrocarbons (PAHs) was determined in the chemical-activated luciferase expression (CALUX) assay, using two exposure times (6 and 24 h), in order to reflect the metabolization of PAHs. AhR-inducing potencies of PAHs were expressed as induction equivalency factors (IEFs) relative to benzo[ a ]pyrene and 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). In 24 h exposure assay, the highest IEFs were found for benzo[ k ]fluoranthene, dibenzo[ a , h ]anthracene and dibenzo[ a , k ]fluoranthene (approximately three orders of magnitude lower than TCDD) followed by dibenzo[ a , j ]anthracene, benzo[ j ]fluoranthene, indeno[1,2,3- cd ]pyrene, and naphtho[2,3- a ]pyrene. The 6 h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24 h exposure (generally by two orders of magnitude), probably due to the high rate of PAH metabolism. The strongest AhR inducers showed IEFs approaching that of TCDD. Several PAHs, including some strong mutagens, such as dibenzo[ a , l ]pyrene, cyclopenta[ cd ]pyrene, and benzo[ a ]perylene, elicited only partial agonist activity. Calculation of IEFs based on EC25 values and/or 6 h exposure data is suggested as an alternative approach to estimation of toxic potencies of PAHs with high metabolic rates and/or the weak AhR agonists. The IEFs, together with the recently reported relative mutagenic potencies of PAHs [Mutat. Res. 371 (1996) 123; Mutat. Res. 446 (1999) 1] were combined with data on concentrations of PAHs in extracts of model environmental samples (river sediments) to calculate AhR-mediated induction equivalents and mutagenic equivalents. The highest AhR-mediated induction equivalents were found for benzo[ k ]fluoranthene and benzo[ j ]fluoranthene, followed by indeno[1,2,3- cd ]pyrene, dibenzo[ a , h ]anthracene, benzo[ a ]pyrene, dibenzo[ a , j ]anthracene, chrysene, and benzo[ b ]fluoranthene. High mutagenic equivalents in the river sediments were found for benzo[ a ]pyrene, dibenzo[ a , e ]pyrene, and naphtho[2,3- a ]pyrene and to a lesser extent also for benzo[ a ]anthracene, benzo[ b ]fluoranthene, indeno[1,2,3- cd ]pyrene, benzo[ j ]fluoranthene, dibenzo[ a , e ]fluoranthene and dibenzo[ a , i ]pyrene. These data illustrate that AhR-mediated activity of PAHs, including the highly mutagenic compounds, occurring in the environment but not routinely monitored, could significantly contribute to their adverse effects.

We investigated whether and how could various modulators of arachidonic acid metabolism affect apoptosis induced by tumour necrosis factor-alpha (TNF-alpha) in human myeloid leukaemia HL-60 cells. These included arachinonyltrifluoromethyl ketone (AACOCF3; cytosolic phospholipase A2 inhibitor), indomethacin (cyclooxygenase inhibitor), MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethyl propanoic acid; 5-lipoxygenase-activating protein inhibitor), nordihydroguaiaretic acid (general lipoxygenase inhibitor), and arachidonic acid itself. Incubation of HL-60 cells with nordihydroguaiaretic acid resulted in apoptosis and it was characterised by mitochondria membrane depolarisation, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. Indomethacin and nordihydroguaiaretic acid synergistically potentiated TNF-alpha-induced apoptosis, while arachidonic acid, AACOCF3 and MK-886 did not modulate its effects. Furthermore, indomethacin potentiated apoptosis in cells treated with a differentiating agent, all-trans retinoic acid, which induces resistance to TNF-alpha. However, the observed effects were probably not associated either with the cyclooxygenase- or lipoxygenase-dependent activities of indomethacin and nordihydroguaiaretic acid, respectively. Since indomethacin may reportedly activate peroxisome proliferator-activated receptors (PPARs), the effects of specific ligands of PPARs on apoptosis were studied as well. It was found that selective PPARs ligands had no effects on TNF-alpha-induced apoptosis. The findings suggest that arachidonic acid metabolism does not play a key role in regulation of apoptosis induced by TNF-alpha in the present model. Nevertheless, our data raise the possibility that indomethacin could potentially be used to improve the treatment of human myeloid leukaemia.