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Archives of Environmental Contamination and Toxicology (v.34, #3)
Peroxidase-Catalyzed Oxidation of 2,4,6-Trichlorophenol by F. W. Wiese; H. C. Chang; R. V. Lloyd; J. P. Freeman; V. M. Samokyszyn (pp. 217-222).
2,4,6-Trichlorophenol (TCP) is an environmental contaminant that is toxic, mutagenic, and carcinogenic. We have investigated peroxidase-catalyzed oxidation of TCP as an alternative pathway of TCP bioactivation using horseradish peroxidase (HRP) as a model peroxidase. TCP was shown to function as a reducing substarte for HRP as evidenced by TCP-dependent, HRP-catalyzed reduction of 5-phenyl-4-penten-1-yl hydroperoxide (PPHP) to its corresponding alcohol. In addition, TCP was shown to undergo hydroperoxide (H2O2, ethyl hydroperoxide, or PPHP)-dependent metabolism as evidenced by electronic absorption spectroscopic analysis of reaction mixtures. A single major product was detected by reverse phase HPLC and was identified as 2,6-dichloro-1,4-benzoquinone (2,6-dichloro-2,5-cyclohexadiene-1,4-dione, CAS no. 697-91-6) on the basis of electronic absorption spectroscopy, mass spectrometry, and cochromatography with synthetic standard. In addition, HRP-catalyzed oxidation of TCP yielded EPR-detectable phenoxyl radical intermediates whose EPR spectrum consisted of a 1:2:1 triplet characterized by proton hyperfine coupling constants aH(3,5)= 2.35 gauss. Mechanisms for the hydroperoxide-dependent, HRP-catalyzed oxidation of TCP are proposed that are consistent with these results.
Macrophage Secretory Function Is Enhanced by Low Doses of Tributyltin-Oxide (TBTO), But Not Tributyltin-Chloride (TBTCl) by D. H. Kergosien; C. D. Rice (pp. 223-228).
Numerous studies suggest that tributyltin (TBT) is a potent immunotoxicant in nontarget organisms with lymphoid atrophy being a hallmark response. Two of the most common formulations of TBT are bis (tri-n-butyl)-tin oxide (TBTO) and tri-n-butyl-tin chloride (TBTCl). Most of studies investigating TBT-related immunotoxicity have used relatively high doses of both compounds, but little is known about the effects of very low doses. In addition, no studies have directly compared the effects of both formulations on immune function(s). We exposed female B6C3F1 mice to a single dose of TBTO or TBTCl at 0.3, 3.0, 30 mM/kg or corn oil as a carrier control. Forty-eight h later mice received a 4% solution of thioglycolate intraperitoneally to elicit peritoneal macrophages. Ninety-six h later macrophages were harvested and stimulated with a mixture of gamma-interferon (IFN-γ) and lipopolysaccharide (LPS). Nitric oxide (NO), tumor necrosis factor-α (TNF-α), transforming growth factor beta-1 (TGF-β1), and phorbol ester-stimulated oxidative burst activity were then measured. Nitric oxide and TNF-α production were significantly elevated in the 0.3 and 3.0 mM TBTO/kg–treated groups but not in those treated by TBTCl. Background TNF-α production (without stimulation) was also elevated at these two doses but suppressed in TBTCl-treated animals. Oxidative burst activity was elevated at 0.3 mM TBTO/kg but not by TBTCl. TGF-β1 production was not altered by either treatment, nor were body wts and organ-body wt ratios. To further evaluate the difference between the effects of TBTO and TBTCl on macrophage function, the in vitro toxicity of the two was determined using elicited peritoneal macrophages from untreated mice. Following a 24-h exposure to increasing concentrations of TBTO or TBTCl, functional viability was evaluated using the MTT assay. There were no differences between the two compounds in terms of treatment-related viability except that at the very highest concentrations (10−6 M) TBTO was more toxic than TBTCl.
In Vitro Cytotoxicity of BTEX Metabolites in Hela Cells by Y. Shen (pp. 229-234).
Fuel leakage from underground storage tanks is a major source of groundwater contamination. Although the toxicity of regulated compounds such as benzene, toluene, ethylbenzene, and xylene (BTEX) are well recognized, the cytotoxicity of their metabolites has not been studied extensively. In this study, Hela cells, propagated at 37°C in an atmosphere of 5% CO2–95% air, served as a target for evaluation of cytotoxicity of BTEX metabolites 3-methylcatechol, 4-methylcatechol, 4-hydroxybenzoic acid, and 4-hydroxy-3-methoxybenzoic acid. The cells were exposed to different concentrations of the metabolites, which subsequently showed inhibition of cell growth and produced dose-related decreases in cell viability and cell protein content. The BTEX metabolites affected the levels of the polyamines spermidine, spermine, and putrescine, which are known to be important in cell proliferation. The cytotoxic effects for these BTEX metabolites to Hela cells were 3-methylcatechol > 4-methylcatechol > 4-hydroxy-3-methoxybenzoic acid > 4-hydroxybenzoic acid.
Photoinduced Toxicity of PAHs and Alkylated PAHs to a Marine Infaunal Amphipod (Rhepoxynius abronius) by B. L. Boese; J. O. Lamberson; R. C. Swartz; R. Ozretich; F. Cole (pp. 235-240).
The marine infaunal amphipod Rhepoxynius abronius was exposed in standard 10-day toxicity tests to sediments contaminated with parent or alkylated PAHs. After exposures, mortalities (LC50 values) and the ability to rebury in control sediment (EC50 values) were determined. Survivors of these initial toxicity tests were then exposed to UV radiation in an environmental growth chamber for 1 h. The differences between EC50 values before and after UV exposure were used to assess the phototoxicity of the bioaccumulated contaminants. Contaminants with HOMO-LUMO gap energies between 7.2 and 7.7 eV produced up to an order-of-magnitude increase in toxicity with UV exposure. The strength of phototoxic responses within this HOMO-LUMO gap range varied with contaminants such that compounds with the lowest water solubilities appeared to be relatively less phototoxic. This suggests that these compounds were not taken up in sufficient quantities to produce a strong phototoxic response and points out the need to measure tissue residues in phototoxicity experiments. In general, these results support the HOMO-LUMO gap model of photoinduced toxicity.
Investigating the Potential Impacts of Chlorophenols on the Lake Baikal (Siberia, Russia) Food Web by Employing Daphnia Grazing Bioassays and a Chlorella Growth Bioassay by J. E. Gokcen (pp. 241-247).
A grazing bioassay was employed to assess the impacts of chlorophenols on Daphnia magna and Daphnia pulex. The effects of two chlorophenols, pentachlorophenol (PCP) and 4-chlorophenol, were investigated at concentrations of 0.001, 0.01, and 0.1 mg · L−1 over a 96-h period. All tests were conducted in water from the southern basin of Lake Baikal (Siberia). For D. magna, grazing rates were significantly depressed after exposure to 0.001 mg · L−1 of PCP for 48 h or to 0.01 mg · L−1 of 4-chlorophenol for 96 h. However, neither chemical continued to depress filtering rates as either dose or time increased, thus effective concentrations (EC50s) could not be determined. This prevents the use of this bioassay as a tool for assessing exposure to chlorophenols, but it is still useful in that it provides insight into potential ecological effects. In the case of D. pulex, depressed rates were also found at 0.001 mg · L−1 of PCP after 48 h; due to problems with the control, no conclusions were drawn for the effect of 4-chlorophenol on this species. The growth rates of Daphnia's prey, Chlorella vulgaris, were also investigated in the presence of these chemicals; no observable effects were found at any concentration during the 96-h period, implying that ecosystem effects may be limited to higher trophic levels.
Quantitative Structure–Activity Relationships and Volume Fraction Analysis for Nonpolar Narcotic Chemicals to the Australian Cladoceran Ceriodaphnia cf. dubia by R. M. Rose; M. St. J. Warne; R. P. Lim (pp. 248-252).
The toxicity of eleven nonpolar narcotic chemicals to the cladoceran Ceriodaphnia cf. dubia was determined. C. cf. dubia was found to be approximately four times more sensitive to these narcotic chemicals than Daphnia magna tested under virtually identical conditions. The toxicity data were also used to develop and validate quantitative structure–activity relationships (QSARs) using a range of physicochemical properties of the chemicals. The three best QSARs, based on octanol–water partition coefficients and two lipid–water partition coefficients, were able to explain 98% of the variation in toxicity. The mean absolute percentage errors between the predicted and experimental EC50 values for these three QSARs were 17.3%, 20.6%, 24.6%. Neither the critical concentration (CC) nor the critical volume (CV) hypotheses validly modeled the toxicity data when octanol–water and triolein–water partition coefficients were used although the CV hypothesis was the better of the two. When a phospholipid–water partition coefficient was used the CV hypothesis was valid. The mean toxic membrane volume fraction of 0.48 × 10−2 m3/m3 derived in this study agreed with published values for nonpolar narcotics and supports the use of this property to determine the mode of action of chemicals.
HSP60 as a Potential Biomarker of Toxic Stress in the Nematode Plectus acuminatus by J. E. Kammenga; M. S. J. Arts; W. J. M. Oude-Breuil (pp. 253-258).
The induction of heat shock proteins (HSPs) in the nematode Plectus acuminatus (Nematoda; Plectidae) was studied following exposure to heat, copper, and cadmium. Mini two-dimensional polyacrylamide gel electrophoresis was used for protein separation and poly- and monoclonal antibodies raised against specific HSPs in various organisms were used to detect specific HSPs. Both HSP60 and HSP70 could be identified after exposure of nematodes to heat stress, indicating the broad crossreactivity among species to the antibodies used. The monoclonal antibody LK-2 was selected for further investigation with the HSP60 response to heavy metals. The induction of HSP60 was related to increased concentrations of cadmium and copper. For copper, the induction of HSP60 was three orders of magnitude more sensitive than was the EC20 for reproduction. For cadmium, HSP60 induction was one order of magnitude more sensitive. The results point out that HSP60 induction occurred at concentration levels that are realistic for the field situation. It is therefore suggested that HSP60 may be suitable as a potential biomarker to toxicant stress in P. acuminatus.
Determining Toxicity Trends in the Ozonation of Synthetic Dye Wastewaters Using the Nematode Caenorhabditis elegans by D. R. Hitchcock; S. E. Law; J. Wu; P. L. Williams (pp. 259-264).
The nematode Caenorhabditis elegans was used in 72-h toxicity tests to evaluate the influence of ozonation on the toxicity of three synthetic azo dye wastewaters (two reactive dyes and one acid-based dye). The two reactive dye wastewaters contained high concentrations of NaCl (89–112 g/L) in addition to potentially toxic dye components. To determine the contribution of NaCl to toxicity, simulated dye wastewater samples with and without NaCl were tested. Samples were collected at various times during ozonation (t = 0, 8, 32, 64 min); nematodes were exposed to the samples for 72 h. The influence of ozonation on toxicity varied between dye wastewater types. For the acid-based dye wastewater, toxicity increased as duration of ozonation increased. For the reactive dyes without NaCl, toxicity did not appear to be influenced by ozonation. For the reactive dyes with NaCl, mortality was 100% with or without ozonation. Range-finding experiments with NaCl in water and NaCl in dye wastewaters suggested an additive toxic interaction between NaCl and the dyes in wastewater to the nematodes. The duration of ozonation for acid-based dyes and the relatively high NaCl concentrations for the reactive dyes appear to influence effluent toxicity in the ozonated dye wastewaters.
Acute Toxicity of Potassium to the Adult Zebra Mussel Dreissena polymorpha by P. J. Wildridge; R. G. Werner; F. G. Doherty; E. F. Neuhauser (pp. 265-270).
The acute toxicity of potassium (K+) to adult zebra mussels, Dreissena polymorpha, and the efficacy of using K+ to enhance the toxicity of a commercial biocide was examined. Mussels, 15–20 mm in total shell length, collected from Lake Ontario, were exposed to static concentrations of K+ for 3, 6, 12, and 24 h, and to a sublethal concentration of K+ prior to and during exposure to Clam-Trol® CT-2 for 6, 12, and 24 h. Tests were conducted at ambient lake temperatures of 12°C and 22°C and mussels were subjected to a 96 h recovery period. Valve closure was inhibited in mussels exposed to sublethal as well as lethal concentrations of K+, resulting in mussels that were nonresponsive to tactile stimulation. The median effective concentration (ED50) of K+ to induce nonresponsive mussels increased as the length of the recovery period was extended from 24 to 96 h, indicating that some nonresponsive mussels were capable of recovering 96 h after exposure to the K+ treatments. A recovery period duration of 96 h was critical in assessing mortality in mussels exposed to high K+ levels and the use of tactile stimulation to test for valve responsiveness was insufficient to identify mortality. The 24 h median lethal concentration (LC50) of K+ at 22°C (400 mg/L) was found to be sixfold higher than the LC50 reported by other investigators utilizing shorter recovery periods. The LC50 of the biocide to mussels treated with K+ was not reduced, suggesting that the use of K+ to inhibit valve closure may not be useful in methods to control mussel infestations.
Molluscicidal and Anti-AChE Activity of Tertiary Mixtures of Pesticides by A. M. Tripathi; R. A. Agarwal (pp. 271-274).
We studied the toxicity and in vivo inhibition of acetylcholinesterase (AChE) by the organophosphate Nuvan (dichlorvos); Nuvan mixed with a mixed function oxidase inhibitor, piperonyl butoxide (PB); Nuvan with a pyrethroid Decis (deltamethrin); and a tertiary mixture of Nuvan, PB, and Decis in the snail Lymnaea acuminata. Nuvan was highly toxic to the snail and was a strong inhibitor of AChE activity. Both PB and Decis act synergistically with Nuvan when given together in a 1:5 and 1:46 ratio, respectively. When a tertiary mixture of Nuvan, Decis, and PB was given in a 1:46:5 ratio, the toxicity of the mixture was higher in comparision to the binary mixture of Nuvan + PB or Nuvan + Decis.
Biochemical Effects of Didecyldimethylammonium Chloride (DDAC) Exposure and Osmoregulatory Stress on Juvenile Coho Salmon, Oncorhynchus kisutch by B. D. Johnston; J. M. Seubert; C. J. Kennedy (pp. 275-279).
The effects of a seawater challenge on coho salmon, Oncorhynchus kisutch, previously exposed to didecyldimethylammonium chloride (DDAC) were examined. In one experiment, salmon were exposed to three sublethal concentrations of DDAC over three durations followed by a 24-h seawater challenge in a computer-controlled, intermittent-flow respirometer to measure effects on several biochemical variables. After a 144-h dose, plasma cortisol, glucose, and gill Na+/K+-ATPase activity were significantly increased at a nominal DDAC concentration of 0.2 mg/L. In the second experiment, animals were exposed to five different concentrations for 24 h followed by a 24-h seawater challenge. Plasma cortisol was significantly increased at the highest exposure concentration (0.75 mg/L). Plasma Na+ was significantly elevated at exposure concentrations of 0.3, 0.5, 0.65, and 0.75 mg/L. Gill Na+/K+-ATPase activity was significantly reduced at exposure concentrations of 0.65 mg/L and 0.75 mg/L. The use of the seawater challenge to demonstrate sublethal physiological stress and impaired osmoregulatory capacity in coho salmon smolts is relevant to salmonid life history in terms of the animal's transition from freshwater to seawater during its seaward migration.
Dietary and Aqueous Exposure of Finfish to Organochlorine Compounds: A Case Study by J. Hellou; D. Mackay; J. H. Banoub (pp. 280-288).
The level of organochlorine compounds was determined in whole capelin, Mallotus villosus, and compared to concentrations determined in tissues of yellowtail flounder, Pseudopleuronectes ferruginea, that fed on capelin for 2 years while maintained in tanks. Capelin represent part of the diet of offshore yellowtail flounder, however, they come to the beaches to spawn and were collected inshore for the feeding experiment. Therefore, inshore–offshore capelin concentrations were compared to investigate differences, while variables such as fish weight and lipid content were examined to give a better view of the range of contaminants concentrations in capelin. During two years, weekly exposure of flounder was to 148 L of water, as opposed to a dietary intake of 1 g of capelin. Although the level of contaminants was only measured in capelin, it can be estimated for the water, using results obtained on the level of contaminants in sediments obtained in a different study. According to our calculations, levels of contaminants were three to 20 times higher from the aqueous compared to the dietary uptake of inshore flounder, increasing with lower hydrophobicity. Exposure was from less than 10 to up to 100 times lower in expected and/or published results for offshore food and water, respectively. This comparison suggests a major influence of inshore waters on the bioaccumulation of contaminants in some inshore marine species, although the effect of altering the diet of captive finfish can not be disregarded.
Sensitivity of Wild Cotton Rats (Sigmodon hispidus) to the Immunotoxic Effects of Low-Level Arsenic Exposure by M. Savabieasfahani; R. L. Lochmiller; D. P. Rafferty; J. A. Sinclair (pp. 289-296).
Arsenic is a ubiquitous contaminant of many toxic waste sites around the country and experimental animal trials have indicated that arsenic may be immunotoxic to laboratory rodents. Because wild rodents such as the herbivorous cotton rat (Sigmodon hispidus) reside on many of these toxic waste sites, we explored the sensitivity of their immune systems to oral exposures of environmentally relevant concentrations of inorganic arsenic. We exposed adult male cotton rats (n = 36) to either 0 (controls), 5 (low dose), or 10 (high dose) ppm sodium arsenite in drinking water for 6 weeks. Daily food intake decreased in a dose-dependent manner, ranging from an average of 10.03 ± 0.45 in the high-dose group to 11.27 ± 0.42 (SE) g/animal/day in the control group. Mass of testes in the low-dose group increased significantly compared to controls, but there was no difference between the high-dose and control groups. Masses of liver, kidney, adrenals, popliteal lymph nodes, spleen, epididymides, and seminal vesicles and selected hematological parameters were unaffected by arsenic exposure. In vivo cell-mediated immunity, as measured by a phytohemagglutinin-hypersensitivity response to an intradermal challenge, was suppressed 30% in the low-dose group compared to controls; however, responses of those receiving a high dose of arsenic were similar to controls. Arsenic treatment did not have a measurable impact on lymphoproliferative responses of cultured splenocytes to the mitogens Concanavalin A and Pokeweed mitogen, or to the lymphokine interleukin-2. We also observed no impact of low-level arsenic exposure on macrophage phagocytic activity and tumoricidal activity of lymphokine-activated killer cells in vitro. It is possible that malnutrition caused by decreased food intake may eventually lead to atrophy of lymphoid organs and render animals more susceptible to environmental pathogens. However, direct effects of low-level arsenic exposure on immune function of cotton rats was minimal (a moderate depression in the in vivo cell-mediated immunity assay) and may not be clinically relevant with regard to susceptibility to disease in the wild.
Urinary Excretion of Arsenic Species After Exposure to Arsenic Present in Drinking Water by P. Kurttio; H. Komulainen; E. Hakala; H. Kahelin; J. Pekkanen (pp. 297-305).
The water from some drilled wells in southwest Finland contains high arsenic concentrations (min–max: 17–980 μg/L). We analyzed inorganic arsenic (As-i) and organic arsenic (monomethylarsonate [MMA] and dimethylarsinate [DMA]) species in urine and conducted a clinical examination of current users (n = 35) and ex-users (n = 12) of such wells. Ex-users had ceased to use the water from the wells 2–4 months previously. Urinary arsenic species were also analyzed from persons whose drinking water contained less than 1 μg/L of arsenic (controls, n = 9). The geometric means of the concentrations of total arsenic in urine were 58 μg/L for current users, 17 μg/L for ex-users, and 5 μg/L for controls. The excreted arsenic was associated with the calculated arsenic doses, and on average 63% of the ingested arsenic dose was excreted in urine. The ratios of MMA/DMA and As-i/As-tot (As-tot = As-i + MMA + DMA) in urine tended to be lower among the current users and in the higher exposure levels than in controls, suggesting that As-i was better methylated in current users. However, the differences were mainly explained by age; older persons were better methylators of inorganic arsenic than younger individuals. The arsenic content of hair correlated well with the past and chronic arsenic exposure; an increase of 10 μg/L in the arsenic concentration of the drinking water or an increase of 10–20 μg/day of the arsenic exposure corresponded to a 0.1 mg/kg increase in hair arsenic. The individuals were interviewed and complained of muscle cramps, mainly in the legs, and this was associated with elevated arsenic exposure. The present study demonstrates that arsenic methylation has no threshold at these exposure levels.
Effects of Xenoestrogenic Environmental Pollutants on the Proliferation of a Human Breast Cancer Cell Line (MCF-7) by A. Blom; E. Ekman; A. Johannisson; L. Norrgren; M. Pesonen (pp. 306-310).
A human breast cancer cell line (MCF-7) was used to develop an in vitro screening assay for the detection of xenoestrogenic environmental pollutants. MCF-7 cells were cultured in DMEM containing 5% fetal bovine serum (FBS). An estrogenic response was defined as an increase in the frequency of proliferating MCF-7 cells, and was measured using a thymidine analog, bromodeoxyuridine, and flow cytometry. Di-2-ethylhexyl phthalate (DEHP) and 4-n-nonylphenol (4-n-NP) were used as model chemicals. The proliferation rate of S-phase cells after 24 h of exposure to various concentrations of 17β-estradiol and to model compounds was compared with a positive and a negative control, containing 1 nM 17β-estradiol and 0.1% ethanol, respectively. DEHP and 4-n-NP increased the frequency of proliferating MCF-7 cells in a dose-dependent manner. The lowest concentration that significantly increased the proliferation of MCF-7 cells was 10 μM for DEHP and 1 μM for 4-n-NP.The results showed that the assay is accurate and quick to perform. It may prove a valuable tool for screening potential estrogen-mimicking environmental pollutants.