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Impairment of humoral immune responses in mice exposed to nitrogen dioxide and ozone mixtures.

The relationship between immune defense mechanisms and environmental pollutants remains unknown because of uncertainty about the effects of combined or mixed pollutants. To investigate whether exposure to toxic gas mixtures change the effect of a single gas exposure on immune function, BALB/c mice were continuously exposed to 4.0 ppm nitrogen dioxide (NO2), 0.8 ppm ozone (O3), or the mixture of NO2 plus O3 for 3, 7, 14, and 56 days. Organ weights (lung, thymus, and spleen) and antibody responses to sheep red blood cells (SRBC), and to DNP-Ficoll were measured immediately after the exposure. Lung weights in mice exposed to O3 or the mixture were increased significantly in all exposure periods. The weights of thymus and spleen in mice exposed for 3, 7, and 14 days to the mixture were decreased. O3 exposure for 56 days showed significant decreases of the weights of both organs. Antibody response to SRBC in mice exposed for 3, 7, and 14 days to O3 or the mixture was markedly suppressed, but exposure to the mixture for 56 days did not show the suppression of anti-SRBC antibody response. No differences in anti-DNP antibody response between exposed and control mice were observed, except those exposed to O3 or the mixture for 14 days. These results suggest that mixed gas exposures variously modify the effects of a single gas exposure on antibody production in mice.

Air Pollutants↗

Toxicological evaluation of chemical mixtures.

This paper addresses major developments in the safety evaluation of chemical mixtures during the past 15 years, reviews today's state of the art of mixture toxicology, and discusses challenges ahead. Well-thought-out tailor-made mechanistic and empirical designs for studying the toxicity of mixtures have gradually substituted trial-and-error approaches, improving the insight into the testability of joint action and interaction of constituents of mixtures. The acquired knowledge has successfully been used to evaluate the safety of combined exposures and complex mixtures such as, for example, the atmosphere at hazardous waste sites, drinking water disinfection by-products, natural flavouring complexes, and the combined intake of food additives. To consolidate the scientific foundation of mixture toxicology, studies are in progress to revisit the biological concepts and mathematics underlying formulas for low-dose extrapolation and risk assessment of chemical mixtures. Conspicuous developments include the production of new computer programs applicable to mixture research (CombiTool, BioMol, Reaction Network Modelling), the application of functional genomics and proteomics to mixture studies, the use of nano-optochemical sensors for in vivo imaging of physiological processes in cells, and the application of optical sensor micro- and nano-arrays for complex sample analysis. Clearly, the input of theoretical biologists, biomathematicians and bioengineers in mixture toxicology is essential for the development of this challenging branch of toxicology into a scientific subdiscipline of full value.

Air Pollutants↗

Comparative toxicity of cadmium, zinc, and mixtures of cadmium and zinc to daphnids.

Investigations were conducted to determine acute (48-h) effects of cadmium and zinc presented individually and in combination on Ceriodaphnia dubia, Daphnia magna, Daphnia ambigua, and Daphnia pulex. Toxicity tests were conducted with single metals to determine lethal effects concentrations (lethal concentrations predicted for a given percent [x] of a population, LCx value). These were used to derive metal combinations that spanned a range of effects and included mixtures of LC15, LC50, and LC85 values calculated for each metal and species. In single-metal tests, 48-h LC50 values ranged from 0.09 to 0.9 micromol/L and 4 to 12.54 micromol/L for cadmium and zinc, respectively. For each metal, D. magna was most tolerant and showed a different pattern of response from all others as determined by slope of concentration-response curves. In the combined metal treatments, all daphnids showed a similar pattern of response when LC15 concentrations were combined. This trend continued with few exceptions when LC15 concentrations of cadmium were combined with LC50 or LC85 values for zinc. However, when this treatment was reversed (LC15, zinc + LC50 or LC85, cadmium), responses of all species except D. magna indicated less-than-additive effects. For C. dubia, a near complete reduction in toxicity was observed when the LC15 for zinc was combined with LC85 for cadmium. Multimetal tests with D. magna did not differ from additive. Collectively, these studies suggest that D. magna may not be representative of other cladocerans.

Animals↗

Chemical contaminants and their effects in fish and wildlife from the industrial zone of Sumgayit, Republic of Azerbaijan.

Sediment from a wetland adjacent to an industrial wastewater treatment plant in Sumgayit contained concentrations of total PAHs, total PCBs, aldrin, biphenyl, chlordane, DDT, mercury, beta-endosulfan, heptachlor, alpha-hexacyclohexane (alpha-HCH), gamma-HCH, and several individual PAH congeners that were elevated relative to published sediment quality guidelines. Chemical analyses of tissues from European pond turtles (Emys orbicularis) had increased levels of many of the same chemicals including aldrin, chlordane, heptachlor, alpha-HCH, total PCBs, total PAHs, and mercury, compared to reference turtles. In addition, turtle tissues contained elevated levels of DDD, hexachlorobenzene (HCB), and pentachlorobenzene that were not elevated in the sediment sample. Some differences were observed in contaminant levels between European pond turtles and Caspian turtles (Mauremys caspica) taken from the ponds in Sumgayit. Salmonella/microsome mutagenicity assays on pond sediments were negative or weakly positive. Micronuclei in European pond turtles were statistically correlated with tissue levels of mercury, heptachlor, DDD, HCB, and trans-nonachlor. Microcosm experiments using Russian sturgeon (Acipenser gueldenstaedtii) showed a positive dose-response relationship between exposure to suspended contaminated pond sediment and acute toxicity. Chemical and biological assays used in this study show the industrial area of Sumgayit is heavily contaminated with a complex mixture of toxic pollutants. Exposure to contaminated sediments produced acute effects in Russian sturgeon, but genotoxic effects appear to be slight.

Animals↗

Aquatic toxicity of four alkylphenols (3-tert-butylphenol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol) and their binary mixtures to microbes, invertebrates, and fish.

The acute and chronic toxicity of four simple alkylphenols with butyl and propyl substitutions was evaluated with aquatic microbes, invertebrates, and fish. These alkylphenols-3-tert-butylphenol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol-have been detected in various environmental media, but their impact on aquatic fauna has seldom been evaluated. Relative susceptibility to each phenolic varied by test species. The marine bacterium Vibrio fischeri was the most susceptible to the alkylphenols, up to 3 orders of magnitude more sensitive than species of higher trophic levels. For 4-isopropylphenol, the 5-min Microtox EC(50) value was 0.01 mg/L, whereas the EC(50) for Ceriodaphnia after a 48-h exposure was 10.1 mg/L. Notable differences in sensitivity to the alkylphenols was also observed with the Microtox assay: 4-isopropylphenol was > 200 times more toxic to V. fischeri than was 2-isopropylphenol (EC(50) = 2.72 mg/L). For V. fischeri, the mixture toxicity of the alkylphenols was additive in nature and was predicted by a concentration addition model. The energy of the lowest unoccupied molecular orbital (ELUMO) explained the observed toxicity of the individual alkylphenols to V. fischeri (r(2) = 0.92, p < 0.05). These results suggest that the mode of action of polar narcotic alkylphenols to V. fischeri is different than that of other test organisms, possibly because of the differences in the cell structure of the prokaryotic V. fischeri.

Animals↗

Assessment of alcohol ethoxylate surfactants and fatty alcohols mixtures in river sediments and prospective risk assessment.

A feasible and relatively readily available analytical method was adapted for the assessment of alcohol ethoxylates (AE) and fatty alcohols (FA) in sediments. This study illustrates the simultaneous measurement of 38 of 114 possible alcohol ethoxylate ethoxymers (AE) and fatty alcohols (FA) found in commercially important AE products. We predicted toxicity for all identified fractions, as well as the total mixture toxicity, relative to three exposure scenarios via sewage treatment plants (STP) for these widely used chemicals in consumer products and hence generate a preliminary environmental risk screening for AE and FA in sediments. The method is based on derivatization of solvent or solid-phase extracts with 2-fluoro-N-methylpyridinium p-toluenesulfonate (Pyr+). The derivatized extracts were analyzed with liquid chromatography/mass spectrometry (LC/MS) operating in the positive ion electrospray mode. The extraction efficiency of AE and FA in three different sediments of varying composition was evaluated with spike-recovery studies, ranging from 64% to 80%. The detection limits for individual ethoxymers typically ranged from 1 to 5 ngg(-1)on a dry weight basis. The mean limit of detection (LOD) was 6 ngg(-1)and the median LOD was 3 ngg(-1). AE and FA in sediments were found to be stable for two weeks if preserved with 3% (v/v) formalin and stored at 4-6( composite function)C. Based on equilibrium partitioning, background concentrations of AE and FA were predicted to be below concentrations known to elicit chronically toxic effects. Total worst case mixture toxicities for all AE ethoxymers combined with FA were predicted to result in a risk quotient less than 0.6. Activated sludge treatment (STP) significantly reduced the release of total AE and FA by four-fold, suggesting that the total mixture risk quotient would be < 0.15 for sediment dependent organisms.

Alcohols↗

[Correction of nitrogen metabolism in rats with toxic hepatitis by parenteral administration of an amino acid mixture].

The nitrogen metabolism in rats with toxic affection of the liver caused by administration of CCl4 was studied as affected by the amino acid mixture moriamine S-2. It is established that with toxic hepatitis the nitrogen metabolism is sharply disturbed, especially during protein deficiency. The following evidences for this fact: a rise in the depth of the nitrogen negative balance, an increase in the intensity of amine nitrogen and ammonia excretion with urea as well as an increase in the amount of amine nitrogen in blood and tissues with a simultaneous decrease in the content of protein. The parenteral administration of the amino acid mixture for eight days to the animals with a toxic affection of the liver is more effective when nitrogen preparation is combined with the group B vitamin, vitamin C, insulin, nerobolyl and sirepar.

Amino Acids↗

Developmental toxicity of D-002 (a mixture of aliphatic primary alcohols) in rats and rabbits.

D-002 is a mixture of higher aliphatic alcohols, primarily isolated and purified from beeswax, that has moderate anti-inflammatory and effective anti-ulcer activity. In the present study the developmental toxicity in Sprague-Dawley rats and New Zealand White rabbits was evaluated. Doses of 100, 320 and 1000 mg kg(-1) were administered once daily by gavage to pregnant females on gestation days 6-15 (rats) or 6-18 (rabbits). Reproductive indices were determined and foetuses were examined for external, visceral and skeletal malformations. There was no indication of embryotoxicity, fetotoxicity or teratogenicity in any of the treated groups. It is concluded that D-002 is devoid of teratogenic or embryotoxic effects in these strains.

Animals↗

Purification and characterization of defensins from cystic fibrosis sputum.

OBJECTIVE AND DESIGN: Cystic fibrosis (CF) sputum contains large numbers of neutrophils whose most abundant granule proteins are defensins. Within phagolysosomes, defensins kill microbes; however, extracellular defensins can be toxic to human cells. To begin to explore the possibility that defensins damage CF airways, this study examines the concentration and properties of defensins in CF sputum. MATERIALS: As a source of biological material in which to assay levels of defensins, purulent sputum was collected from persons with CF hospitalized with exacerbations of bronchitis. Purulent CF sputum was also a source of material for purification of defensins. METHODS: Defensin concentration in the cell-free component of CF sputum was measured by immunoassay. Defensins acid-extracted from sputum were purified by Sephacryl S-200 gel filtration chromatography and reverse phase-high pressure liquid chromatography (HPLC). Toxicity of CF defensins was tested by incubating the purified defensins with a line of CF tracheal cells cultured in serum five medium. RESULTS: In 5 patients with CF, sputum defensin levels ranged from 300 to > 1600 micrograms/ml, higher than levels previously reported in any human secretion or fluid and greatly exceeding concentrations toxic to mammalian cells in vitro. HPCL-purified CF sputum defensins were pure as judged by acid urea-PAGE and N-terminal sequencing, which revealed a mixture of defensins-1, -2 and -3 at ratios of approximately 4:2:1. At > 10 micrograms/ml the purified mixture was toxic for a line of CF tracheal cells cultured in serum-free medium, as judged by reductions in cell numbers and increased permeability to trypan blue. CONCLUSIONS: This study suggests that defensins in CF sputum are intact and sufficiently abundant that they may damage airway epithelium.

Blood Proteins↗

Identification and quantification of toxic chemicals by use of Escherichia coli carrying lux genes fused to stress promoters.

The luxCDABE bioluminescence genes of the Vibrio fischeri lux system have been used as a reporter system for different stress and regulatory promoters of Escherichia coli. Selected E. coli strains carrying lux genes fused to different promoters were exposed to various toxic chemicals, and the recorded luminescence was used for the characterization of the biologic signature of each compound. Analysis of these data with the aid of a proper algorithm allowed quantitative and qualitative assessment of toxic chemicals. Of the 25 tested chemicals, 23 were identified by this novel strategy in a 3-h procedure. This system can also be adapted for the identification of simple mixtures of toxic agents when the biologic signatures of the individual compounds are known. This biologic recognition strategy also provides a tool for evaluating the degree of similarity between the modes of action of different toxic agents.

Escherichia coli↗

The joint toxic action of perchloroethylene with benzene or toluene in rats.

The joint toxic action of mixtures of perchloroethylene with benzene or toluene was tested at five dose levels for each of six binary combinations with 20 rats per dose. LD50 data with 95% fiducial limits are reported for each mixture and tests for additive joint toxicity were made. The results indicate that the mixtures of perchloroethylene and benzene gave values for the LD50 which were slightly less than additive while perchloroethylene and toluene mixtures had LD50 values which could not be predicted by an additive model.

Animals↗

Fish consumption advisories: toward a unified, scientifically credible approach.

A model is proposed for fish consumption advisories based on consensus-derived risk assessment values for common contaminants in fish and the latest risk assessment methods. The model accounts in part for the expected toxicity to mixtures of chemicals, the underlying uncertainties in the health and exposure data, and the amount of contaminated fish consumed. Application of the model to a larger number of chemicals is possible. Noncancer toxicity is used as an example, but this model is applicable for risks from cancer as well. A second related model is proposed that is useful for comparing potential risks among sites (e.g., rivers and lakes).

Animals↗

Optimization of an exogenous metabolic activation system for FETAX. I. Post-isolation rat liver microsome mixtures.

The developmental toxicity of cyclophosphamide, coumarin, 2-acetyl-aminofluorine (2-AAF), and trichloroethylene (TCE) was assessed with Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to each test material for 96-h in two separate static-renewal tests with and without the presence of five differently induced exogenous metabolic activation systems (MAS). The MAS consisted of Aroclor 1254- (Aroclor 1254 MAS), isoniazid- (INH MAS), phenobarbital- (PB MAS), or beta-naphthoflavone- (beta-NF MAS), or a post-isolation mixture (mixed MAS) of INH-, PB-, and beta-NF-induced rat liver microsomes. Addition of the Aroclor 1254 MAS bioactivated cyclophosphamide, coumarin, 2-AAF, but not TCE. Addition of the PB MAS bioactivated cyclophosphamide, weakly bioactivated coumarin and 2-AAF, but had no effect on TCE developmental toxicity. The beta-NF MAS bioactivated coumarin and 2-AAF, weakly bioactivated cyclophosphamide, but did not alter the developmental toxicity of TCE. Addition of the INH-induced MAS only bioactivated TCE, whereas the post-isolation mixed MAS bioactivated each test material. Based on LC50 and EC50 (malformation) values, embryo growth, and types and severity of induced malformations, each test material was developmentally toxic. Use of post-microsome isolation mixtures from differentially induced rat livers increased the efficacy of the exogenous MAS routinely used by FETAX.

2-Acetylaminofluorene↗

Ultrastructural alterations in the liver and intestine of carp Cyprinus carpio induced orally by ultra-low doses of endosulfan.

In order to elucidate the importance of food-borne chemical contamination in fish, cytological and ultrastructural alterations in hepatocytes and enterocytes of common carp Cyprinus carpio L. exposed for 5 wk to 0.5 microgram endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9a-hexahydro-6,9-methano-2,4,3-benzo- dioxyanthiepin-3-oxide) kg-1 food dry weight, equivalent to an ultra-low dosis of 15 ng kg-1 fish d-1, were investigated by means of light and electron microscopy. Observations on liver alterations were quantified by morphometric analysis. Livers show enlargement of the nucleolus, increase in number and size of both Golgi fields and rough endoplasmic reticulum (ER) lamellae, as well as proliferation of peroxisomes and lysosomes. Taken together, these alterations represent the morphological equivalent of a general stimulation of hepatic metabolism. Proliferation of the smooth ER is indicative of the onset of biotransformation processes under the influence of food-borne endosulfan. Further pathological processes in the liver were evident by glycogen and lipid depletion, invasion of phagocytic macrophages, and accumulation of myelinated bodies in endothelial cells of hepatic sinusoids. In the intestinal tract, exposure to endosulfan is associated with a complete lack of chylomicrons in the epithelial lining, which indicates disturbance of intestinal absorption. The reaction of the gut epithelium also included considerable distension of the intercellular space and an elevated number of lysosomal inclusions in enterocytes. An increased rate of mucous cell precursors was detectable, and macrophages were numerous. Results are consistent with endosulfan resorption by the intestinal epithelium and the coexistence of gut and liver ultrastructural changes at extremely low doses. Thus, the substantiation of pathological alterations in organs sequentially in contact with toxicants appears useful as a biomarker of pollutant exposure and effect. With regard to a chemical spill into the Rhine river at Basel, Switzerland, in November 1986, endosulfan, as a component of the mixture of toxic substances, may well have contributed to the overall toxicity of the chemicals released during the accident and the subsequent fish kill, less as a toxicant in itself than as a stimulant for the toxicity of other xenobiotics.

Administration, Oral↗

Some thoughts on a possible regulatory approach at EEC level on the classification and labeling of dangerous preparations.

At the present time no European directives on classification, packaging, and labeling exist which cover all dangerous preparations of chemicals. To develop such a directive, the Commission of the European Economic Community services, at the request of and in close collaboration with experts of the member states, has conceived a method for evaluating the toxicity of mixtures of chemicals. This method consists of (a) the characterization of each constituent by means of an index and (b) the classification of preparations for each route of administration on the basis of their toxicity. The index is a figure representing the degree of dilution needed to render the substance not dangerous in the sense of the EEC Directives on the classification of dangerous substances; the highest index among those obtained for acute and chronic toxicity is taken to characterize the substances. An indication of toxicity is obtained by applying the simple equation sigma (P X I), where P is the percentage and I is the index of each constituent. The most stringent classification among those obtained for the three routes of administration is taken for the final classification of the preparation on the basis of its acute and chronic toxicity. Other toxicological effects such as irritation, corrosion, sensitization, mutagenesis, teratogenesis, and carcinogenesis are not considered.

Animals↗

trans-Nonachlor and cis-nonachlor toxicity in Sprague-Dawley rats: comparison with technical chlordane.

cis-Nonachlor and trans-nonachlor are bioaccumulating components of the pesticide chlordane, which can be detected in various environmental biota and in humans. Existing studies have focused on the potential adverse health effects of the parent chlordane mixture. Comparable toxicity data are nonexistent for individual chlordane constituents such as trans-nonachlor, cis-nonachlor, or oxychlordane, which are among the most common chlordane-related environmental contaminants and tissue residues. In this study, rats were administered cis-nonachlor, trans-nonachlor, or technical chlordane by gavage for 28 days at doses of 0.25 to 25 mg/kg body weight. Residue analyses indicated that trans-nonachlor accumulation in adipose was greater than cis-nonachlor when rats were administered each chemical under identical conditions of dose and exposure. For all test chemicals, the major metabolite oxychlordane accumulated in adipose tissue. Adipose tissue residue levels of all test chemicals and the major metabolite were higher in female rats. The liver was a target organ in male and female rats, indicated by increased liver weight and histopathological changes consistent with microsomal enzyme induction. Hepatic changes were most pronounced in rats treated with trans-nonachlor. Elevated kidney weights and depressed organic ion transport were observed in males treated with trans-nonachlor and chlordane. Although in general, changes in target organs and clinical chemistry endpoints were similar for all 3 test chemicals, the approximate toxicity ranking from most to least toxic was trans-nonachlor > technical chlordane > cis-nonachlor.

Animals↗

Arsenic-cadmium interaction in rats: toxic effects in the heart and tissue metal shifts.

Previously, we had shown that arsenic interacts with cadmium in rats; our results showed that the toxicity of a mixture of arsenic + cadmium cannot be predicted by the toxic mechanisms of the individual components. In this paper, we present further evidence about the interaction of arsenic and cadmium in rats. The results were: arsenic modified the 24 h-LD50 value of cadmium more clearly than cadmium did with the one of arsenic; based on the LD50 values, the mixtures we studied were more toxic than either metal alone. With single doses (As 10 mg/kg, Cd 2.6 mg/kg, and As 10 mg/kg + Cd 2.6 mg/kg) the mixture As + Cd was more toxic than each metal. At these doses, cadmium significantly induces the levels of glutathione, metallothionein, and lipid peroxidation in heart tissue, as compared to a saline group of rats. Arsenic incremented glutathione and lipid peroxidation at higher values than those obtained with cadmium. The mixture of As + Cd behaved as arsenic in the induction of lipid peroxidation and glutathione and like cadmium in metallothionein induction. Finally, rats treated with As + Cd had less Cd in liver than animals treated only with cadmium, and more As in heart tissue than rats treated only with arsenic. Our results give further evidence about the arsenic-cadmium interaction in rats, demonstrate the utility of employing different biomarkers in the study of chemical mixtures and indicate that heart tissue is affected not only by the mixture of As + Cd, but also by either metal alone.

Animals↗

Toxic effects of unresolved complex mixtures of aromatic hydrocarbons accumulated by mussels, Mytilus edulis, from contaminated field sites.

Exposure of marine mussels (Mytilus edulis) to an unresolved complex mixture (UCM) of aromatic hydrocarbons isolated from a crude oil has been shown to reduce their feeding rate by 40%. The present study was undertaken to determine whether UCMs bioaccumulated by mussels in the field are also toxic. The feeding rate of mussels derived from polluted sites increased when they were placed in clean water, pointing to a loss of toxic agents from the tissues. At the end of the depuration period, water in which mussels from an oil-polluted site had been held contained a UCM. Steam-distillation extracts of the tissues of mussels taken from several polluted sites were shown to be highly toxic to the feeding activity of juvenile mussels. The tissues of mussels from these sites contained UCMs. Nontoxic steam distillates from clean mussels did not. Steam-distillation extracts of mussels from an oil-polluted site were fractionated by normal-phase high-performance liquid chromatography. A fraction, largely comprising a "monoaromatic" UCM, reduced the feeding rate of juvenile mussels by 70%. Two later-eluting fractions containing aromatic UCMs also produced smaller depressions in feeding rate. These results support our contention that some aromatic UCM hydrocarbons constitute a forgotten pollutant burden in the marine environment.

Animals↗