Kirby C. Donnelly

Acute and genetic toxicity of municipal landfill leachate

The large number of municipal solid waste (MSW) landfills and the many hazardous materials which they contain pose a serious environmental threat to our groundwater reserves. The present study was conducted to assess the environmental hazards that four MSW landfill leachates pose to the groundwater. Genetic toxicities of the landfill leachate and groundwater samples were assessed using the Salmonella/microsome (Ames test) mutagenicity bioassay, the Bacillus subtilis DNA repair bioassay, and the diploid Aspergillus nidulans chromosome damage bioassay. Acute toxicities of the leachate samples were assessed using the Microtox test. The leachate and groundwater samples were also analyzed for organic constituents using gas chromatography and gas chromatography/mass spectrometry. The chemical data were used to calculate the estimated cumulative cancer risk for each sample. All leachate samples were acutely toxic, and three of the four leachate samples were genetically toxic. Two of the four leachate samples and the groundwater sample contained concentrations of USEPA priority pollutants in excess of promulgated standards for potable water. Two of the leachates had mean estimated cumulative cancer risks on the same order of magnitude (10−4) as leachates from co-disposal and hazardous waste landfills. The use of a battery of acute and genetic toxicity bioassays, chemical analysis, and an estimated cancer risk calculation resulted in evidence that MSW landfill leachates are as acutely and chronically toxic as co-disposal and hazardous waste landfill leachates.

Nondietary ingestion of pesticides by children in an agricultural community on the US/Mexico border: preliminary results.

An environmental measurement and correlation study of nondietary ingestion of pesticides was carried out in a colonia in south Texas. The purpose of the study was to evaluate young childrens exposure to environmental levels of organophosphate (OP) pesticides in the household. Samples were collected to measure levels of OP pesticides in housedust and on childrens hands. These, in turn, were compared to levels of OP pesticide metabolites in urine. A total of 52 children, 25 boys and 27 girls, participated in the spring and summer of 2000. The children were 7–53 months of age at the time of recruitment. Univariate and multivariate regression analyses were carried out using SAS statistical software. Seventy-six percent of housedust samples and 50% of hand rinse samples contained OP pesticides. All urine samples had at least one metabolite and over 95% had at least two metabolites above the limit of detection (LOD). Total OP loadings in the housedust ranged from nondetectable (nd) to 78.03 nmol/100 cm2 (mean=0.15 nmol/100 cm2; median=0.07 nmol/100 cm2); total OP loadings on the childrens hands ranged from nd to 13.40 nmol/100 cm2 (mean=1.21 nmol/100 cm2; median=1.41 nmol/100 cm2), and creatinine corrected urinary levels (nmol/mol creatinine) of total OP metabolites ranged from 3.2 to 257 nmol/mol creatinine (mean=42.6; median 27.4 nmol/mol creatinine). Urinary metabolites were inversely associated with the age of the child (in months) with the parameter estimate (pe)=−2.11, P=0.0070, and 95% confidence interval −3.60 to −0.61. The multivariate analysis observed a weak association between concentrations of OP pesticides in housedust, loadings in housedust, and concentration on hands, hand surface area, and urinary levels of OP metabolites. However, hand loadings of OP pesticides were more strongly associated (r2=0.28; P=0.0156) with urinary levels of OP metabolites (pe=6.39; 95% CI 0.98–11.80). This studys preliminary findings suggest that surface loadings of pesticides, on hands, are more highly correlated with urinary bioassays and, therefore, may be more useful for estimation of exposure in epidemiologic studies than levels of pesticides in housedust.

Porous organoclay composite for the sorption of polycyclic aromatic hydrocarbons and pentachlorophenol from groundwater

Complex mixtures of hazardous chemicals such as polycyclic aromatic hydrocarbons (PAHs) in contaminated soil and groundwater can have severe and long-lasting effects on health. The evidence that these contaminants can cause adverse health effects in animals and humans is rapidly expanding. The frequent and wide-spread occurrence of PAHs in groundwater makes appropriate intervention strategies for their remediation highly desirable. The core objective of this research was to assess the ability of a clay-based composite to sorb and remove toxic contaminants from groundwater at a wood-preserving chemical waste site. Treatment efficiencies were evaluated using either effluent from an oil-water separator (OWS) or a bioreactor (B2). The effluent water from these units was passed through fixed bed columns containing either an organoclay composite or granular activated carbon. The sorbent columns were placed in-line using existing sampling ports at the effluent of the OWS or B2. Individual one-liter samples of treated and untreated effluent were collected in Kimax bottles over the course of 78 h (total of 50 samples). Subsequently each sample was extracted by solid phase extraction methodology, and pentachlorophenol (PCP) and PAH concentrations were quantitated via GC/MS. Columns containing porous organoclay composite, i.e. sand-immobilized cetylpyridinium-exchanged low-pH montmorillonite clay (CP/LPHM), were shown to reduce the contaminant load from the OWS effluent stream by 97%. The concentrations of benzo[a]pyrene (BaP) and PCP were considerably reduced (i.e. >99%). An effluent stream from the bioreactor was also filtered through columns packed with composite or an equivalent amount of GAC. Although the composite reduced the majority of contaminants (including BaP and PCP), it was less effective in diminishing the levels of lower ring versus higher ring PAHs. Conversely, GAC was more effective in removing the lower ring PAHs, except for naphthalene and PCP. The effectiveness of sorption of PCP from the OWS effluent by the composite was confirmed using a PCP-sensitive adult hydra bioassay previously described in our laboratory. The findings of this initial study have delineated differences between CP/LPHM and GAC for groundwater remediation, and suggest that GAC (instead of sand) as the solid support for organoclay may be more effective for the treatment of contaminated groundwater under field conditions than GAC or CP/LPHM alone. Further work is ongoing to confirm this conclusion.

Neurotoxicity induced in differentiated SK-N-SH-SY5Y human neuroblastoma cells by organophosphorus compounds.

Organophosphorus (OP) compounds used as insecticides and chemical warfare agents are known to cause potent neurotoxic effects in humans and animals. Organophosphorus-induced delayed neuropathy (OPIDN) is currently thought to result from inhibition of neurotoxic esterase (NTE), but the actual molecular and cellular events leading to the development of OPIDN have not been characterized. This investigation examined the effects of OP compounds on the SY5Y human neuroblastoma cells at the cellular level to further characterize cellular targets of OP neurotoxicity. Mipafox and paraoxon were used as OP models that respectively do and do not induce OPIDN. Mipafox (0.05 mM) significantly decreased neurite length in SY5Y cells differentiated with nerve growth factor (NGF) while paraoxon at the same concentration had no effect when evaluated after each of three 4-day developmental windows during which cells were treated daily with OP or vehicle. In contrast, paraoxon but not mipafox altered intracellular calcium ion levels ([Ca(2+)](i)), as seen in three types of experiments. First, immediately following the addition of a single high concentration of OP to the culture, paraoxon caused a transient increase in [Ca(2+)](i), while mipafox up to 2 mM had no effect. Paraoxon hydrolysis products could also increase intracellular Ca(2+) levels, although the pattern of rise was different than it appeared immediately after paraoxon administration. Second, repeated low-level paraoxon treatment (0.05 mM/day for 4 days) decreased basal [Ca(2+)](i) in NGF-differentiated cells, though mipafox had no effect. Third, carbachol, a muscarinic acetylcholine receptor agonist, transiently increased [Ca(2+)](i) in differentiated cells, an affect attenuated by 4-day pretreatment with paraoxon (0.05 mM/day), but not by pretreatment with mipafox. These results indicate that the decrease in neurite extension that resulted from mipafox treatment was not caused by a disruption of Ca(2+) homeostasis. The effects of OPs that cause or do not cause OPIDN were clearly distinguishable, not only by their effects on neurite length, but also by their effects on Ca(2+) homeostasis in differentiated SY5Y cells.

Children's mouthing and food-handling behavior in an agricultural community on the US/Mexico border

Childrens mouthing and food-handling activities were measured during a study of nondietary ingestion of pesticides in a south Texas community. Mouthing data on 52 children, ranging in age from 7 to 53 months, were collected using questionnaires and videotaping. Data on childrens play and hand-washing habits were also collected. Children were grouped into four age categories: infants (7–12 months), 1-year-olds (13–24 months), 2-year-olds (25–36 months) and preschoolers (37–53 months). The frequency and type of events prompting hand washing did not vary by age category except for hand washing after using the bathroom; this increased with increasing age category. Reported contact with grass and dirt also increased with increasing age category. The median hourly hand-to-mouth frequency for the four age groups ranged from 9.9 to 19.4, with 2-year-olds having the lowest frequency and preschoolers having the highest. The median hourly object to mouth frequency ranged from 5.5 to 18.1 across the four age categories; the frequency decreased as age increased (adjusted R2=0.179; P=0.003). The median hourly hand-to-food frequency for the four age groups ranged from 10.0 to 16.1, with the highest frequency being observed in the 1-year-olds. Hand-to-mouth frequency was associated with food contact frequency, particularly for children over 12 months of age (adjusted R2=0.291; P=0.002). The frequency and duration of hand-to-mouth, object-to-mouth and food-handling behaviors were all greater indoors than outdoors. Infants were more likely to remain indoors than children in other age groups. The time children spent playing on the floor decreased with increasing age (adjusted R2=0.096; P=0.031). Parental assessment was correlated with hand-to-mouth activity but not with object-to-mouth activity. The highest combined (hand and object) mouthing rates were observed among infants, suggesting that this age group has the greatest potential for exposure to environmental toxins.

Chromosomal aberrations in native small mammals (Peromyscus leucopus and Sigmodon hispidus) at a petrochemical waste disposal site: I. Standard karyology.

Examination of standard metaphase chromosome preparations was employed to evaluate the use of resident small mammals as indicators of environmental mutagenesis. Small mammals of two species, (Peromyscus leucopus andSigmodon hispidus) were trapped over a two-year period at a locality polluted with a complex mixture of petrochemical waste products, heavy metals, and PCBs (polychlorinated biphenyls), and from two uncontaminated localities. Significant differences in levels of chromosomal aberrations between animals collected at the contaminated site and the uncontaminated sites were clearly indicated. Increases in lesions per cell and aberrant cells per individual were shown for both species at the contaminated site compared to the control sites. Levels of chromosomal aberrations were not different between the two control sites, however. This study suggests that cytogenetic analysis of resident small mammals is a feasible test model for assessment of environmental mutagenesis.

Evolutionary toxicology: population-level effects of chronic contaminant exposure on the marsh frogs (Rana ridibunda) of Azerbaijan.

We used molecular methods and population genetic analyses to study the effects of chronic contaminant exposure in marsh frogs from Sumgayit, Azerbaijan. Marsh frogs inhabiting wetlands in Sumgayit are exposed to complex mixtures of chemical contaminants, including petroleum products, pesticides, heavy metals, and many other industrial chemicals. Previous results documented elevated estimates of genetic damage in marsh frogs from the two most heavily contaminated sites. Based on mitochondrial DNA (mtDNA) control region sequence data, the Sumgayit region has reduced levels of genetic diversity, likely due to environmental degradation. The Sumgayit region also acts as an ecological sink, with levels of gene flow into the region exceeding gene flow out of the region. Additionally, localized mtDNA heteroplasmy and diversity patterns suggest that one of the most severely contaminated sites in Sumgayit is acting as a source of new mutations resulting from an increased mutation rate. This study provides an integrated method for assessing the cumulative population impacts of chronic contaminant exposure by studying both population genetic and evolutionary effects.

Biomonitoring of exposure in farmworker studies.

Although biomonitoring has been used in many occupational and environmental health and exposure studies, we are only beginning to understand the complexities and uncertainties involved with the biomonitoring process—from study design, to sample collection, to chemical analysis—and with interpreting the resulting data. We present an overview of concepts that should be considered when using biomonitoring or biomonitoring data, assess the current status of biomonitoring, and detail potential advancements in the field that may improve our ability to both collect and interpret biomonitoring data. We discuss issues such as the appropriateness of biomonitoring for a given study, the sampling time frame, temporal variability in biological measurements to nonpersistent chemicals, and the complex issues surrounding data interpretation. In addition, we provide recommendations to improve the utility of biomonitoring in farmworker studies.

Pregnane X receptor protects HepG2 cells from BaP-induced DNA damage.

Pregnane X receptor (PXR) is a nuclear receptor that coordinately regulates transcriptional expression of both phase I and phase II metabolizing enzymes. PXR plays an important role in the pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics. Metabolism of benzo[a]pyrene (BaP), a well-established carcinogen and ubiquitous environmental contaminant, can result in either detoxification or bioactivation to its genotoxic forms. Therefore, PXR could modulate the genotoxicity of BaP by changing the balance of the metabolic pathways in favor of BaP detoxification. To examine the role of PXR in BaP genotoxicity, BaP-DNA adduct formation was measured by 32P-postlabeling in BaP-treated parental HepG2 cells and human PXR-transfected HepG2 cells. The presence of transfected PXR significantly reduced the level of adducts relative to parental cells by 50-65% (p < 0.001), demonstrating that PXR protects liver cells from genotoxicity induced by exposure to BaP. To analyze potential PXR-regulated detoxification pathways in liver cells, a panel of genes involved in phase I and phase II metabolism and excretion was surveyed with real-time quantitative reverse transcription PCR. The messenger RNA levels of CYP1A2, GSTA1, GSTA2, GSTM1, UGT1A6, and BCRP (ABCG2) were significantly higher in cells overexpressing PXR, independent of exposure to BaP. In addition, the total GST enzymatic activity, which favors the metabolic detoxification of BaP, was significantly increased by the presence of PXR (p < 0.001), independent of BaP exposure. Taken together, these results suggest that PXR plays an important role in protection against DNA damage by polycyclic aromatic hydrocarbons (PAHs) such as BaP, and that these protective effects may be through a coordinated regulation of genes involved in xenobiotic metabolism.

Relationship between genotoxicity, mutagenicity, and fish community structure in a contaminated stream

Genotoxic responses (chromosomal damage, DNA strandbreakage) of redbreast sunfish (Lepomis auritis)populations exposed to industrial effluent andmutagenicity of the associated sediments weredetermined in order to compare them to changes incommunity structure. Data were collected from areference stream and East Fork Poplar Creek (EFPC), afirst-order stream which originates on the grounds ofthe Department of Energy Y-12 Plant at Oak Ridge, TN. This stream is contaminated with mercury, PCBs, andnumerous other compounds. Previous studies have shownthat sediment contaminant concentrations, as well asphysiological biomarker responses of the local fishpopulations, are highest at the headwaters of EFPC anddecrease with increasing distance from the DOEfacility as contaminant loading decreases. Chromosomal damage was measured by flow cytometry – asreflected by variation in cellular DNA content – andstrand breakage was determined by agarose gelelectrophoresis using blood as the source of DNA. Mutagenicity was determined by theSalmonella/microsome assay using organic solventextracts of sediment surface samples. Community levelresponses included community diversity and percentpollution-tolerant species. Biomarker responses andmutagenicity were found to be highest at theheadwaters of EFPC, and tended to decrease withincreasing distance from the effluent. In general,biomarker responses appeared to be correlated withmutagenicity of the sediment, and both of theserelated to fish community disturbance and level ofstream contamination. Because responses at severallevels of biological organization show similarpatterns of downstream effects, this suggests thatthere may be a causal relationship betweencontamination and biological effects.