Biochemical screening of highly toxic aromatic contaminants in river sediment and comparison of sensitivity of biological model systems (original) (raw)
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Environmental Toxicology and Chemistry, 2002
On the basis of a new fractionation method combined with in vitro ethoxyresorufin-O-deethylase (EROD) induction in a rainbow trout liver cell line (RTL-W1) and chemical analysis, halogenated aromatic hydrocarbons with dioxin-like activity were identified in a sediment extract from Bitterfeld, Germany. The fractionation method allowed a separation of different nonplanar and coplanar polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs) with different degrees of chlorination. The dioxin-like activity at the investigated site could be quantitatively assigned to PCDD/Fs. Both PCBs and PCNs could be excluded as the cause of the measured effects on the basis of the fractionation procedure and bioanalytical results. Thus, the method allowed the chemical analysis to focus on PCDD/Fs, with significant reduction of the analytical expense. The EROD-induction potency of sediment-extract fractions was quantified, and toxicants were confirmed by the application of induction equivalent quantities on the basis of fixed-effect-level concentrations that exhibit 15% of the maximum induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin. This approach was designed to minimize methodological limitations due to superimposing inhibitory effects.
Environmental Toxicology and Chemistry, 2001
Extracts of sediment samples collected from the Morava River and its tributaries (Czech Republic) were examined for mutagenic, dioxin-like, and estrogenic activities. Moreover, the human leukemic HL-60 cell line was tested as a potential model for the detection of effects of environmental contaminants on cell proliferation and differentiation processes. Analytical data indicate that the sediments were contaminated predominantly with polycyclic aromatic hydrocarbons (PAHs) and phthalate esters. The sums of concentrations of 16 U.S. Environmental Protection Agency priority PAHs ranged from 0.8 to 13.2 g/g and those of phthalates reached up to 3,000 ng/g, while only low levels of chlorinated hydrocarbons were found. The main goal of the present study was to determine effects of PAH prevalence on in vitro bioassays, with special emphasis on dioxin-like activity. The dioxin-like activity was tested using a reporter gene assay based on chemical-activated luciferase expression (the CALUX assay). Significant dioxinlike activity (2.6-40.1 g/g benzo[a]pyrene equivalents and 5.9-48.2 ng/g 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents) was detected in all samples, and the results obtained with various exposure times or with both crude and PAH-deprived extracts indicate that the response was probably caused almost exclusively by the presence of high concentrations of PAHs. This corresponds with results of chemical analyses and indicates that various exposure times would allow a discrimination between dioxin-like activities of persistent compounds and easily metabolized aryl hydrocarbon (Ah) receptor inducers. Only sediment extracts containing the highest concentrations of PAHs were mutagenic, as determined by the umu assay. Estrogenic activity was found in several samples (4.75-22.61 pg/g estradiol equivalents) using cells stably transfected with an estrogen-responsive element linked to a luciferase promoter. Noncytotoxic doses of extracts had no effects on HL-60 cell proliferation, while two of the tested crude extracts significantly enhanced their all-trans retinoic acid-induced differentiation. These activities were not associated with phthalate esters and/or PAHs. Our results indicate that cellular and biochemical in vitro assays based on various specific modes of action may yield data complementary to results of mutagenicity tests and that they could be useful in environmental risk assessment. High levels of PAHs are apparently associated with dioxin-like and mutagenic activities rather than with estrogenic activity.
Archives of Toxicology, 1996
Cytochrome P450-dependent 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-dealkylase (PROD) and 7-ethoxycoumarin Odeethylase (ECOD) activities in 14-day-old chick embryo livers were determined 24 h after pretreatment with selected widespread aromatic environmental contaminants, including polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), hexachlorobenzene, and dialkylesters of phthalic acid, and compared with the inducing potencies of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and the coplanar and mono-o-chlorinated PCBs. The effects of other model inducers, i.e. phenobarbital and pyrazole, were also examined. Specificity of EROD induction was estimated with regard to contaminants frequently present in environmental samples and dose-response curves for EROD induction were determined. A strong induction (comparable with that by mono-o-chlorinated biphenyl treatment) by dibenzo[a,h]anthracene, benzo[k]fluoranthene or benzo[b]fluoranthene was found, but the maximal level of EROD activity inducible by TCDD was not achieved, partly due to the high toxicity of the tested PAHs. 3-Methylcholanthrene showed moderate inducing potencies; benz[a]anthracene, benzo[a]pyrene, chrysene and 2,2,3,4,4,5-hexachlorobiphenyl appeared to be weak inducers. Other PAHs and PCBs tested, as well as hexachlorobenzene, dialkyl phthalates, phenobarbital and pyrazole had no marked effects on the EROD level. ECOD activities were increased non-specifically by TCDD, 3-methylcholanthrene, hexachlorobenzene and phenobarbital. A significant enhancement of PROD activity by TCDD and related inducers was M. Machala ( ) · I. Svoboda · K. Nezveda
Aquatic Toxicology, 1990
Levels of total cytochrome P-450, of specific P-450 (determined immunologically with MAb 1-12-3 and referred to as P-450E) and ethoxyresorufin O-deethylase (EROD) were elevated in intestine and liver microsomes of spot (Leiostomus xanthurus) collected from the Elizabeth River, a polycyclic aromatic hydrocarbon (PAH) contaminated tributary of Chesapeake Bay. Fish were collected over a sediment PAH concentration gradient that ranged from 9 to 96000 pg PAH/kg dry sediment. Intestinal P-450E was near the lower limits of detection in fish collected at the relatively clean sites but was elevated 80-to 100-fold in fish collected from contaminated sites. Intestinal EROD activity exhibited a similar trend. Liver P-450E and associated EROD activity was detectable in all samples and was induced approximately eight-fold at the most heavily contaminated site. Despite the sensitivity of the intestine to PAH inducing agents, intestinal P-450E levels did not correlate well with sediment PAH, whereas liver P-450E did. Instead, the intestinal enzyme was induced to similar and high levels at all contaminated sites. The results suggest that the intestine plays an important role in the absorption and metabolism of dietary PAH and/or other PAHtype inducing agents and that intestinal P-450E may be a useful indicator of exposure to these compounds via the diet.
Archives of Environmental Contamination and Toxicology, 1996
The bioavailability of soil-bound polycyclic aromatic hydrocarbons (PAHs) for mammalian species was studied with rats fed with a diet containing contaminated soil preparations. The extent of cytochrome P450IA1 (CYP1A1) induction in the liver correlated with the amount of 5-and 6-ring PAHs in the soil samples but not with the total PAH content. Other cytochromes P450 were much less affected by the soil-contaminants. The highest induction of CYP1A1 was obtained with a sample containing 274 mg 5-and 6-ring PAH/kg soil, resulting in a nearly 360-fold increase in the ethoxyresorufin deethylase (EROD) activity. In a semilogarithmic plot, a linear correlation was found between the 5-and 6-ring PAH concentration in the soil and the microsomal CYP1A 1 content. As a model for the action of intestinal fluids, soil samples were extracted by bile acid solution. In these experiments, the selectivity in the solubilization of individual PAHs parallels that of toluene extraction, although the yield is lower than the latter and varies with the soil sample. The bioavailability of PAHs for microorganisms, but not for mammals, was shown to be considerably reduced in the presence of high total organic carbon (TOC) values of the soil samples. This may have implications for decontamination strategies, diminishing the effectiveness of biological decontamination in cases with high TOC values. The data suggest that CYP1A1 induction in rats is a parameter that may be useful in risk assessments of contaminated soils for mammalian species.
Toxicology and Applied Pharmacology, 1997
sediment (Spacie and Hamelink, 1985; Knezovich et al., Route-Specific Cellular Expression of Cytochrome P4501A Bruggeman et al., 1984; Varanasi et al., 1985; Rubin-(CYP1A) in Fish (Fundulus heteroclitus) following Exposure to stein et al., 1984). Although intestine and gill are considered Aqueous and Dietary Benzo[a]pyrene. VAN VELD, P. A., VOGELto be the major routes of toxicant uptake, the relative impor-Similarly, the contribution of contaminated food, water, and Mummichog (Fundulus heteroclitus), an estuarine, teleost fish, sediment to overall dose remains controversial. This inforwere exposed for 456 hr to environmentally relevant concentramation is needed because toxicity is dependent not only on tions of aqueous (10 mg/liter) and dietary (10 mg/g) benzo[a]pyrene dose and duration of exposure, but also on the route by (BP) in static renewal aquaria. Cellular expression of BP-inducible which exposure occurs (Bloomquist, 1992; Driver et al., cytochrome P4501A (CYP1A) was evaluated several times during 1991; Grimmer et al., 1988; Le Curieux et al., 1992; Schurexposure by immunohistochemistry in longitudinal histologic sections of whole fish. CYP1A-associated staining intensities in tissues dak and Randerath, 1989; Xu et al., 1992). were scored by a subjective rating system similar to that used In vertebrates, exposure to diverse xenobiotics results in previously for qualitative information. Exposure to aqueous BP induction of one or more forms of enzymes involved in resulted in high levels of CYP1A-associated immunohistochemical xenobiotic biotransformation (Black and Coon, 1987; Buhler staining in gill pillar cells, heart endothelium, and vascular endoand Williams, 1989). Cytochrome P4501A (CYP1A) forms thelium. Exposure to dietary BP resulted in only mild to moderate are the major oxidative enzymes induced in fish and other staining in these tissues but high-intensity staining in gut mucosal vertebrates by polycyclic aromatic hydrocarbons (PAHs) and epithelium. CYP1A induction in hepatocytes appeared most sensipolyhalogenated aromatic hydrocarbons (PHAHs) (Stegetive to aqueous exposure. Route-specific patterns of CYP1A exman and Hahn, 1994). CYP1A responds to environmental pression were also observed in other cells including gill epithelia, levels of these compounds in a dose-dependent manner and pseudobranch, and skin. Expression of CYP1A in renal tubules and interrenal tissues was not affected by either treatment. Coex-is commonly used in field and laboratory studies to evaluate posure to both aqueous and dietary BP resulted in a pattern of exposure and effects. induction reflecting both routes of exposure. In addition to the
Aquatic Toxicology, 1998
The induction of ethoxyresorufin-o-deethylase (EROD) activity by binary combinations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and seven polychlorinated biphenyls (PCBs) was examined in liver cell lines from rat (H4IIE) and rainbow trout (RTL-W1). The non-ortho PCBs were 77, 81, 126, and 169; the mono-ortho PCBs were 105, 118, and 156. Binary combinations yielded less-than-additive interactions more commonly with RTL-W1 than with H4IIE. In RTL-W1, less-than-additive behaviour was observed at mono-ortho PCB/TCDD ratios of greater than 10000 to 1 and at non-ortho PCB/TCDD ratios of greater than 80 to 1. In H4IIE, less-than-additive interactions were observed at mono-ortho PCB/TCDD ratios of greater than 20000 to 1, but only additive interactions were seen between TCDD and non-ortho PCBs. With both cell lines, greater-than-additive interactions were not found. These results imply that bioassays with these cell lines could lead to lower TCDD-equivalent concentrations than would be determined by chemical analysis.
Determination the Toxicity of Poly Aromatic Hydrocarbons Using HPLC in Diyala River Sediments
Journal of Al-Nahrain University Science, 2014
In this paper sixteen Polycyclic Aromatic Hydrocarbons (PAHs) are estimated in sediments in five locations along Diyala River where entering Baghdad city until flows into Tigris River, (PAHs) are organic compounds that contain two or more fused rings. Some of these compounds are classified as carcinogenic and mutagenic pollutants. Soxhlet technique is used in this work to extract PAHs from sediment samples with using methylene chloride as extraction solvent. High Performance Liquid Chromatography (HPLC) with UV-Vis detector is used for determination of PAHs. According to the results the maximum of total PAHs concentration in the sediment are found in the location No. 4 with1211.22mg/kg in summer and 211312 mg/kg in winter, while the minimum of total PAHs concentration are found in location No. 1 with 44342 mg/kg in summer and 14322 mg/kg in winter. Locations numbers two three and five record values of total PAHs concentration with (141311, 122341 and 011322 mg/kg) respectively in summer and (11340, 122342 and 121311 mg/kg) respectively in winter. Naphthalene, Fluorene, Anthracene and Fluoranthene are the most dominant compounds in summer, while Benzo [k] fluoranthene, Benzo [a] pyrene, Benzo [ghi] perylene, Dibenz [a,h] anthracene and Indino [121cd] pyreneare the least compounds presence in all sites.
Archives of Environmental Contamination and Toxicology, 1997
The H4IIE rat hepatoma cell bioassay has been extensively used to assess the toxic equivalents (TEQs) of complex mixtures of halogenated aromatic hydrocarbons in environmental samples. However, there is often a discrepancy between bioassay induction results and toxic equivalents calculated from chemical analysis of samples; the former generally yield higher bioassay-TEQs. Polynuclear aromatic hydrocarbons (PAHs) are a class of chemicals which can significantly contribute to induction-TEQs. Benzo(a)pyrene (BAP), dibenz(a, h)anthracene (DBA), benz(a)anthracene (BA), benzo(k)fluoranthene (BkF), benzo(b)fluoranthene (BbF), chrysene (Chr), and indeno(1,2,3-c,d) pyrene (IdP) are carcinogenic PAHs found in environmental samples, including oysters collected from Galveston Bay. The induction potency of these PAHs relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was determined individually in rat hepatoma H4IIE cells seeded in 6-well plates, and the induction-derived equivalency factors (EFs) relative to TCDD were 0.000354, 0.00203, 0.000025, 0.00478, 0.00253, 0.00020, 0.0011 for BAP, DBA, BA, BkF, BbF, Chr, and IdP, respectively. Dilutions of a reconstituted PAH mixture containing 23 PAHs (744 to 4466 ng/g total PAHs) with constant percentages of BAP (4.5%), DBA (3.5%), BA (2.4%), BkF (3.7%), BbF (3.5%), Chr (4.7%), and IdP (4.2%) yielded bioassay-derived induction-EQs that ranged from 0.52 to 1.44 ng/g. Oysters exposed in the laboratory to the same PAH mixture for 30 days differentially accumulated the PAHs with time. Bioassay-EQs for these oyster extracts ranged from 0.94 to 5.79 ng/g. These results were similar to the chemically calculated EQs which varied from 0.81 to 3.13 ng/g.