Subsurface microbial communities as a tool for characterizing regional-scale groundwater flow.

Subsurface microbial community distribution patterns are influenced by biogeochemical and groundwater fluxes and may inform hydraulic connections along groundwater-flow paths. This study examined the regional-scale microbial community of the Death Valley Regional Flow System and evaluated whether subsurface communities can be used to identify groundwater-flow paths between recharge and discharge areas. Samples were collected from 36 sites in three groundwater basins: Pahute Mesa-Oasis Valley (PMOV), Ash Meadows (AM), and Alkali Flat-Furnace Creek Ranch (AFFCR). Microbial diversity within and between communities varied by location, and communities were separated into two overall groups that affiliated with the AM and PMOV/AFFCR basins. Network analysis revealed patterns between clusters of common microbes that represented groundwaters with similar geochemical conditions and largely corroborated hydraulic connections between recharge and discharge areas. Null model analyses identified deterministic and stochastic ecological processes contributing to microbial community assemblages. Most communities were more different than expected and governed by dispersal limitation, geochemical differences, or undominating processes. However, certain communities from sites located within or near the Nevada National Security Site were more similar than expected and dominated by homogeneous dispersal or selection. Overall, the (dis)similarities between the microbial communities of DVRFS recharge and discharge areas supported previously documented hydraulic connections between: (1) Spring Mountains and Ash Meadows; (2) Frenchman and Yucca Flat and Amargosa Desert; and (3) Amargosa Desert and Death Valley. However, only a portion of the flow path between Pahute Mesa and Oasis Valley could be supported by microbial community analyses, likely due to well-associated artifacts in samples from the two Oasis Valley sites. This study demonstrates the utility of combining microbial data with hydrologic, geologic, and water-chemistry information to comprehensively characterize groundwater systems, highlighting both strengths and limitations of this approach.

Toxicokinetics of cis- and trans-Permethrin: Influence of Isomer, Maturation, and Sex.

Permethrin exposure of children and adults is widespread in many populations, but knowledge of its relative toxicokinetics (TK) and health risks in immature age groups is lacking. Studies were conducted in rats to determine the influence of immaturity and sex (on plasma and target organ dosimetry of each of the insecticide's 2 isomers, cis- and trans-permethrin [CIS and TRANS]). Postnatal day 15, 21, and 90 (adult), Sprague Dawley rats were orally administered a graduated series of doses of CIS and TRANS in corn oil. Serial sacrifices were conducted over 24 h to obtain plasma, brain, liver, skeletal muscle, and fat profiles of CIS and TRANS. Levels of TRANS decreased relatively rapidly, despite administration of relatively high doses. Concentrations of each isomer in plasma, brain, and other tissues monitored were inversely proportional to the animals' age. The youngest pups exhibited 4-fold higher plasma and brain area under the curves than did adults. Little difference was observed in the TK of CIS or TRANS between adult male and female rats, other than higher initial plasma and liver CIS levels in females. Elevated exposure of the immature brain appears to be instrumental in increased susceptibility to the acute neurotoxicity of high-dose permethrin (Cantalamessa [1993]), but it remains to be established whether age-dependent TK is relevant to long-term, low-level risks.

Plasma Protein and Lipoprotein Binding of Cis- and Trans-Permethrin and Deltamethrin in Adult Humans and Rats.

The majority of residents of the United States, Canada, and Europe are exposed to pyrethroids, the most commonly used class of insecticides. Surprisingly little is known about key aspects of their pharmacokinetics, including their mode of transport in the systemic circulation. This study tested the hypothesis that pyrethroids are transported by both plasma lipoproteins and proteins, similarly to other highly lipophilic environmental contaminants. Other aims were to characterize the binding of representative type I and II pyrethroids, and to compare their binding to rat versus human plasma. Binding of 14C-labeled cis-permethrin (CIS), trans-permethrin (TRANS) and deltamethrin (DLM) to proteins and lipoproteins was measured by sequential extraction of spiked plasma with isooctane, 2-octanol, and acetonitrile. Binding of DLM, CIS, and TRANS to plasma proteins and lipoproteins was linear from 250 to 750 nM; concentrations present in the plasma of orally dosed rats. Binding of DLM to high-density lipoprotein was twice that to low-density lipoprotein. Binding of DLM, CIS, and TRANS was ∼2-fold greater to proteins than to lipoproteins of rat and human plasma; albumin was primarily responsible for protein binding. Higher total binding of each pyrethroid to human (∼90%) than to rat (∼80%) plasma resulted from higher protein binding in human plasma. This was attributable in part to the higher albumin/protein content of human plasma. Rat albumin exhibited lower pyrethroid binding capacity than did human albumin. The results of this investigation indicate that albumin and lipoproteins play a major role in binding and transport of pyrethroids in the systemic circulation of both rats and humans.

Evaluation of Age-Related Pyrethroid Pharmacokinetic Differences in Rats: Physiologically-Based Pharmacokinetic Model Development Using In Vitro Data and In Vitro to In Vivo Extrapolation.

An in vitro to in vivo (IVIVE) extrapolation based-physiologically based pharmacokinetic (PBPK) modeling approach was demonstrated to understand age-related differences in kinetics and how they potentially affect age-related differences in acute neurotoxic effects of pyrethroids. To describe the age-dependent changes in pyrethroid kinetics, it was critical to incorporate age-dependent changes in metabolism into the model. As such, in vitro metabolism data were collected for 3 selected pyrethroids, deltamethrin (DLM), cis-permethrin, and trans-permethrin, using liver microsomes and cytosol, and plasma prepared from immature and adult rats. Resulting metabolism parameters, maximum rate of metabolism (Vmax) and Michaelis-Menten constant (Km), were biologically scaled to respective in vivo parameters for use in the age-specific PBPK model. Then, age-dependent changes in target tissue exposure, i.e., brain Cmax, to a given pyrethroid were simulated across ages using the model. The PBPK model recapitulated in vivo time-course plasma and brain concentrations of the 3 pyrethroids in immature and adult rats following oral administration of both low and high doses of these compounds. A single model structure developed for DLM was able to describe the kinetics of the other 2 pyrethroids when used with compound- and age-specific metabolism parameters, suggesting that one generic model for pyrethroids as a group can be used for early age-sensitivity evaluation if appropriate metabolic parameters are used. This study demonstrated the validity of applying IVIVE-based PBPK modeling to development of age-specific PBPK models for pyrethroids in support of pyrethroid risk assessment of potentially sensitive early age populations in humans.

Age Dependency of Blood-Brain Barrier Penetration by cis- and trans-Permethrin in the Rat.

Permethrin (PER), a type I pyrethroid, is the most widely used insecticide in domestic settings in the United States. The overall objective of this study was to assess the efficiency of the blood-brain barrier (BBB) as an obstacle to the 14C-cis-permethrin (CIS) and 14C-trans-permethrin (TRANS) isomers of PER, and to determine whether its barrier function changes during maturation of the rat. Experiments were conducted to quantify brain uptake of CIS and TRANS in postnatal day 145, 21, and 90 Sprague-Dawley rats. The common carotid artery of anesthetized rats was perfused for 2 or 4 minutes with 1, 10, or 50 µM 14C-CIS or 14C-TRANS in 4% albumin. Brain deposition of each isomer was inversely related to age, with levels in the youngest animals >5 times those in adults. Brain uptake was linear over the 50-fold range of pyrethroid concentrations, indicative of passive, nonsaturable BBB permeation. The extent of uptake of toxicologically relevant concentrations of CIS and TRANS was quite similar. Thus, dissimilar BBB permeation does not contribute to the greater acute neurotoxic potency of CIS, but greater permeability of the immature BBB to CIS and TRANS may contribute to the increased susceptibility of preweanling rodents to the insecticides.

Effect of dose and exposure protocol on the toxicokinetics and first-pass elimination of trichloroethylene and 1,1,1-trichloroethane.

Trichloroethylene (TCE) and 1,1,1-trichloroethane (TRI) are frequent contaminants of drinking water and of groundwater at hazardous waste sites. There is relatively little information on the target organ deposition of TRI, despite its ingestion and common occurrence in humans. An important aim of the study was to delineate and contrast the toxicokinetics (TK) and bioavailability (F) of TRI and its well metabolized congener, TCE. Blood profiles were obtained from male Sprague-Dawley rats given aqueous emulsions of 6 or 48 mg TRI/kg and 10 or 50 mg TCE/kg as an oral bolus (po) or by gastric infusion (gi) over 2 h. TCE exhibited nonlinear TK, with a disproportionate increase in AUC and decrease in clearance and F with increase in dose. TRI exhibited linear TK. F did not vary significantly with TRI dose or dosage regimen. F values were substantially higher for TRI than for the respective TCE groups. TRI was distributed widely to tissues of rats gavaged with 6 mg TRI/kg, with accumulation in fat. This experiment yielded tissue uptake and elimination profiles and in vivo tissue:blood partition coefficients (PCs). Finally, additional rats were given 10 mg/kg of TCE and TRI po, ia and iv, so that first-pass hepatic (FPh) and pulmonary (FPp) elimination could be measured directly. Total and FPh elimination of TCE exceeded that of TRI. TRI, with its higher air:blood PC, exhibited the higher FPp. TCE and TRI, despite several common physical and chemical properties resulting in similar absorption and systemic distribution, displayed dissimilar dosage and dose rate effects on their TK.

Toxicokinetics of Deltamethrin: Dosage Dependency, Vehicle Effects, and Low-Dose Age-Equivalent Dosimetry in Rats.

There is increasing concern that infants and children may be at increased risk of neurological effects of pyrethroids, the most widely used class of insecticide. The objectives of this investigation were to (1) characterize the dose-dependent toxicokinetics (TK) of deltamethrin (DLM) for exposures ranging from environmentally relevant to acutely toxic; (2) determine the influence of an aqueous versus oil vehicle on oral absorption and bioavailability; and (3) determine whether DLM exhibits low-dose, age-equivalent internal dosimetry. Serial arterial plasma samples were obtained for 72 h from adult, male Sprague Dawley rats given 0.05-5.0 mg DLM/kg as an oral bolus in corn oil (CO). DLM exhibited linear, absorption rate-limited TK. Increases in maximum plasma concentration (Cmax) and AUC∘∞ were directly proportional to the dose. Oral bioavailability was quite limited. The vehicle and its volume had modest effect on the rate and extent of systemic absorption in adult rats. Postnatal day (PND) 15, 21, and 90 (adult) rats received 0.10, 0.25, or 0.50 mg DLM/kg orally in CO and were sacrificed periodically for plasma, brain, and liver collection. Age-dependent differences between PND 15 and 90 plasma Cmax and AUC∘24 values progressively diminished as the dose decreased, but there was a lack of low dose age equivalence in these brain and liver dosimeters. Other maturational factors may account for the lack of the low-dose age equivalence in brain and liver. This investigation provides support for the premise that the relatively low metabolic capacity of immature subjects may be adequate to effectively eliminate trace amounts of DLM and other pyrethroids from the plasma.

Gas chromatography/negative chemical ionization mass spectrometry of transfluthrin in rat plasma and brain.

RATIONALE: Transfluthrin is a relatively non-toxic rapid-acting synthetic pyrethroid insecticide. It is widely used in household and hygiene products. A sensitive and accurate bioanalytical method is required for quantification of its concentration in plasma and its potential target organ, the brain for studies to assess its health effects and toxicokinetics in mammals. METHODS: The samples were prepared by liquid-liquid extraction. Gas chromatography mass spectrometry (GC/MS) analysis was performed for the determination of transfluthrin in biological samples with an overall method run time of 15 min. Transfluthrin was quantified using selected-ion monitoring (SIM) in the negative chemical ionization (NCI) mode. Chromatographic separation was achieved using a Zebron® ZB5-MS GC column operating with 1 mL/min constant flow helium. Cis-Permethrin was used as the internal standard. RESULTS: The method was validated to be precise and accurate within the linear range of 1.0-400.0 ng/mL in plasma and 4.0-400.0 ng/mL in brain homogenate, based on a 100 µL sample volume for both matrices. This method was applied to samples following administration of a 10 mg/kg oral dose to male adult rats. The plasma concentrations were observed to be 11.70 ± 5.69 ng/mL and brain concentrations 12.09 ± 3.15 ng/g when measured 2 h post-dose. CONCLUSIONS: A rapid GC/NCI-MS method was demonstrated to be sensitive, specific, precise and accurate for the quantification of transfluthrin in rat plasma and brain. The optimized method was successfully used to quantify the rat plasma and brain concentrations of transfluthrin 2 h after the oral dosing of Sprague-Dawley rats.

Simultaneous determination of cis-permethrin and trans-permethrin in rat plasma and brain tissue using gas chromatography-negative chemical ionization mass spectrometry.

A sensitive method for the simultaneous determination of cis-permethrin (cis-PERM) and trans-permethrin (trans-PERM) in small volumes (100µL) of rat plasma and brain homogenate was developed, using a liquid-liquid extraction for sample preparation and gas chromatography-negative chemical ionization mass spectrometry (GCNCI-MS) for detection. Quantitation of trace levels of the insecticide in small volumes of biological samples is essential to support toxicokinetic studies in small animals. There are currently no validated methods in the literature for determining cis-PERM and trans- PERM in volumes as low as 100µL of rat plasma or brain homogenate. The method provided a linear range of 0.2-150.0ng/mL for analytes in both matrices. The intra- and inter-batch precision (as% relative standard deviation, RSD) and accuracy (as relative error, RE) of the method were better than 20% at the limit of quantitation and better than 15% across the remaining linear range. The validated method was applied in a toxicokinetic study in adult rats with oral dosing of 10mg/kg (cis-PERM) and 100mg/kg (trans-PERM) in corn oil. cis-PERM and trans- PERM were monitored in rat plasma and brain tissue samples for 6h following dosing, and both analytes were detected in all plasma and brain samples.

Brain uptake of deltamethrin in rats as a function of plasma protein binding and blood-brain barrier maturation.

Pyrethroids, including permethrin and deltamethrin (DLM), are very widely used of insecticides. It was hypothesized that lower plasma binding and increased blood-brain barrier (BBB) penetration of DLM in immature rats contribute to the higher brain concentrations of DLM and more pronounced neurotoxicity reported in this age group. The left brain of anesthetized adult rats was perfused for 2min via a carotid artery with 1µM 14C-DLM in: 2-5% human serum albumin (HSA); plasma from adult and 15- and 21-d-old rats; and plasma from human donors of: birth-1 week, 1-4 weeks, 4 weeks-1 year, 1-3 years and adults. The fraction of DLM bound and brain uptake of DLM did not vary significantly with the HSA concentration nor with the age of rat or human plasma donors. One, 10 and 50µM 14C-DLM were perfused into the left-brain of anesthetized adult, 15- and 21-d-old rats. DLM deposition in the brain was linear over this range of concentrations and inversely related to age. The results of this investigation indicate that increased BBB permeability in the youngest rats enhances brain deposition of the insecticide. Plasma protein binding of DLM in immature rats and humans is not sufficiently diminished to impact its brain uptake.

Influence of Maturation on In Vivo Tissue to Plasma Partition Coefficients for Cis- and Trans-Permethrin.

Permethrin, the most widely used household insecticide in the United States, is marketed as a mixture of its cis (CIS) and trans (TRANS) isomers. The major objective of this investigation is to develop and utilize a reliable approach to determine in vivo partition coefficients (PCs) for CIS and TRANS in immature and adult Sprague-Dawley rats. Adult, postnatal day (PND) 21, and PND 15 rats were infused with environmentally relevant concentrations of CIS or TRANS via a subcutaneous osmotic pump for 48 or 72 h. The adult and PND 21 rats also received an oral loading dose. Systemic steady-state or equilibrium was attained in each age group within 72 h of the protocol. CIS and TRANS were both distributed to tissues according to their neutral lipid content, with adipose tissue exhibiting much higher tissue:plasma PCs than skeletal muscle, liver, or brain. Liver:plasma and brain:plasma PCs were consistently at or lower than unity. Tissue:plasma PCs were generally higher for CIS than for TRANS, although the isomers are of comparable lipophilicity. Significantly higher blood levels of CIS apparently saturate plasma binding, resulting in greater tissue deposition of the isomer. CIS and TRANS tissue:plasma PCs were found to be inversely related to the rats' age, although TRANS brain:plasma PCs were comparable in immature and mature animals. These data support the conclusion that age-dependent partitioning is an important determinant of the pharmacokinetics of permethrin. Such partitioning could influence the risk assessment of these insecticides in infants and children when incorporated into physiologically based pharmacokinetic models.

The immature rat as a potential model for chemical risks to children: Ontogeny of selected hepatic P450s.

Concern about potential susceptibilities of infants and children to chemicals has led to the consideration of immature rodents as potential test surrogates. Maturation of some hepatic microsomal cytochrome P450s (CYPs), that participate in metabolic activation of organic solvents and polycyclic aromatic hydrocarbons (PAHs), may differ significantly between humans and rodents. The present investigation was undertaken to delineate the ontogeny of selected hepatic CYPs in male and female Sprague-Dawley (S-D) rats, and to contrast them with developmental profiles in humans. Microsomes were prepared from the liver of sexed and unsexed postnatal day (PND) 1-90 rats, and total CYP450 levels, as well as CYP1A1/2, CYP2E1 and CYP2B1/2 activities and protein, were quantified. CYP1A1/2 and CYP2E1 activity and expression rose rapidly after birth, peaked from PND 21-40/50, and declined substantially to adult values by PND 90. The same ontogenic profiles were manifested when the enzyme activities were expressed per entire liver or liver normalized to body weight. CYP1A1/2 and CYP2E1 activity and protein expression were well correlated. CYP2B1/2 activity peaked abruptly on PND 21 and declined irregularly to adult values. These patterns are in contrast to human CYP1A2 and CYP2E1, which are reported to progressively increase in liver during the first few months to years of life. The three CYP protein developmental profiles were largely gender independent in rats. The immature rat does not appear to be a suitable model for assessing risks posed to infants and children by chemicals metabolically activated by CYP2E1, based on the findings of greater carbon tetrachloride hepatotoxicity in preweanlings and weanlings than in adult animals. Additional studies are required to determine whether immature S-D rats may be used as an animal model for substrates of other CYPs, as total CYP450 levels in the liver progressively rose during maturation, similarly to humans.

Plasma protein binding limits the blood brain barrier permeation of the pyrethroid insecticide, deltamethrin.

Previous pharmacokinetic studies of deltamethrin (DLM) have revealed that brain levels of this highly lipophilic pyrethroid insecticide are only 15-20% of plasma levels. Experiments were performed to assess determinants limiting CNS access including plasma protein binding and the efflux transporter, P-gp. A human brain microvascular endothelial cell line, hCMEC/D3, was utilized as a model in vitro system to evaluate blood-brain barrier (BBB) permeation. Incubation of DLM with a series of human serum albumin (HSA) concentrations showed that unbound (fu) DLM ranged from 80% with 0.01% HSA to ∼20% at the physiologically-relevant 4% HSA. A positive correlation (R=0.987) was seen between fu and cellular uptake. Concentration-dependent uptake of DLM in 0.01% HSA was non-linear and was reduced at 4°C and by the P-gp inhibitor cyclosporine (CSA), indicative of a specific transport process. Cellular accumulation of [(3)H]-paclitaxel, a P-glycoprotein (P-gp) substrate, was increased by CSA but not by DLM, suggesting that DLM is neither a substrate nor an inhibitor of P-gp. The concentration-dependent uptake of DLM from 4% HSA was linear and not significantly impacted by temperature or CSA. In situ brain perfusion studies monitoring brain association of DLM at 0.01% and 4% HSA confirmed the aforementioned in vitro findings. This study demonstrates that brain uptake of DLM under normal physiological conditions appears to be a passive, non-saturable process, limited by the high protein binding of the pyrethroid.

Ontogeny of plasma proteins, albumin and binding of diazepam, cyclosporine, and deltamethrin.

BACKGROUND: To characterize the ontogeny of plasma albumin and total proteins, due to the lack of a comprehensive pediatric database. Secondly, to establish the magnitude and duration of maturational changes in binding of highly-bound drugs/chemicals. METHODS: Anonymized plasma samples from 296 donors were pooled in 6 age brackets from birth to adolescence. Total protein and albumin levels were measured in each age group, as was the age-dependency of plasma binding of diazepam (DZP), cyclosporine (CYC), and deltamethrin (DLM), a pyrethroid insecticide. RESULTS: Plasma levels of albumin and total proteins steadily increased for the first 1-3 y of life. Unbound DZP and CYC fractions were elevated three- to fourfold in neonates, but decreased to adult levels after 1 and 3 y, respectively. Unbound DLM levels exceeded those in adults for just 1 mo. CONCLUSION: Neonates and infants under 1-3 y may be at risk from increased amounts of free drug, when given standard doses of some highly-bound drugs. Pyrethroid insecticides might be anticipated to pose increased risk for 1 mo.

Measurement of plasma protein and lipoprotein binding of pyrethroids.

INTRODUCTION: A simple, reliable procedure was developed to measure binding of pyrethroid insecticides to total proteins and lipoproteins of rat and human plasma. METHODS: The extent of binding of (14)C-labeled deltamethrin (DLM), cis-permethrin (CIS) and trans-permethrin (TRANS) was quantified by a 3-step organic solvent extraction technique. Rat and human plasma samples, containing NaF to inhibit esterases, were spiked with a range of concentrations of each radiolabeled pyrethroid. Protein binding reached equilibrium within ~1h of incubation at 37°C. The samples were extracted in turn with: isooctane to collect the unbound fraction; 2-octanol to extract the lipoprotein-bound fraction; and acetonitrile to obtain the protein-bound fraction. RESULTS: Absolute recoveries of DLM, CIS and TRANS ranged from 86 to 95%. Adherence of these very lipophilic chemicals to glass and plastic was minimized by using silanized glass vials and LoBind® plastic pipettes. The method's ability to distinguish lipoprotein from protein binding was confirmed by experiments with diazepam and cyclosporine, drugs that bind selectively to albumin and lipoproteins, respectively. DISCUSSION: This procedure was effectively utilized for studies of the species-dependence of plasma protein and lipoprotein binding of three pyrethroids for inclusion in physiologically-based pharmacokinetic models of pyrethroids for use in health risk assessments of the insecticides in children and adults.

Determination of deltamethrin in rat plasma and brain using gas chromatography-negative chemical ionization mass spectrometry.

Quantification of the pyrethroid deltamethrin (DLM) in small (100 µL) biological samples from rodents is essential for toxicokinetic studies of trace levels of the insecticide in foods. Such empirical kinetic data are necessary for construction of valid physiologically-based toxicokinetic models. There are no validated methods in the literature for determining deltamethrin in 100 µL plasma and brain samples. Plasma and brain samples were stabilized using sodium fluoride as an esterase inhibitor, and the DLM was extracted by protein precipitation using acetonitrile and phosphoric acid. The samples were vortexed, centrifuged, evaporated to dryness, and reconstituted in toluene prior to injection into a gas chromatograph equipped with a quadrupole mass analyzer. Samples were ionized via electron capture in the negative ion mode using methane, and the molecular ion and fragment ions of DLM were monitored using Selected-Ion Monitoring (SIM) for quantitation and verification of the analyte. Cis-permethrin was used as the internal standard for the method, which was validated according to current US FDA guidelines. Linearity was determined between 0.3 and 1,000 ng/mL, with a limit of detection of 150 pg/mL. The intra- and inter-batch variation for precision (as % relative standard deviation, RSD) and accuracy (as % bias) of the method were better than 20% at the limit of quantitation and better than 15% across the remaining linear range (n=18), with recoveries of 113% and 68% for plasma and brain respectively. Benchtop stability, autosampler stability, and freeze/thaw stability studies of the method (over a 3-day freeze/thaw cycle) were found to be within the acceptance criteria of 20% RSD and bias. This optimized method was applied to the quantitation of DLM in plasma and brain homogenate samples obtained up to 12h after oral dosing of Sprague-Dawley rats with 1mg DLM/kg body weight.

Lack of P-glycoprotein-mediated efflux and the potential involvement of an influx transport process contributing to the intestinal uptake of deltamethrin, cis-permethrin, and trans-permethrin.

The effectiveness and widespread use of pyrethroid insecticides has lead to concerns regarding their safety. Human ingestion of these potentially neurotoxic compounds is typically through hand-to-mouth contact or consumption of contaminated foods. A substantial proportion of ingested pyrethroids are eliminated in feces, suggesting that absorption is limited, possibly by the action of the efflux transporter P-glycoprotein (P-gp). We utilized caco-2 cells as a model system for intestinal enterocytes and qualitatively and quantitatively assessed the transport of deltamethrin (DLM), cis-permethrin (CPM), and trans-permethrin (TPM). Caco-2 cell uptake of the P-gp substrate R6G was increased by the P-gp inhibitors cyclosporine A (CSA) and ritonavir but not by DLM, CPM, and TPM. Unexpectedly, CSA and ritonavir significantly reduced the uptake of DLM, CPM, and TPM. Permeability coefficients (P app) and directional flux of DLM, CPM, or TPM were greater in the absorptive than the secretory (efflux) direction when measured across caco-2 monolayers grown on Transwell inserts. When CSA was applied to the monolayers' apical (AP) side, the AP to basolateral (BL) P app was significantly reduced, with no change in the BL to AP P app. Kinetic analysis demonstrated saturable transport kinetics for all 3 pyrethroids. These findings indicate that the cellular uptake of DLM, CPM, and TPM is not limited by P-gp efflux but undergo absorptive influx transport as a contributing mechanism for cellular uptake. However, the overall P app values for DLM, CPM, and TPM are consistent with the low permeability/low absorption compound mannitol, suggesting limited gastrointestinal absorption potential.

Dominance of epiphytic filamentous Thiothrix spp. on an aquatic macrophyte in a hydrothermal vent flume in Sedge Bay, Yellowstone Lake, Wyoming.

Sublacustrine hydrothermal vents, geysers, and fumaroles impart regions of Yellowstone Lake with distinctive chemical compositions that generate unique freshwater habitats and support diverse microbial life. Some microbial communities within Sedge Bay manifest themselves as accumulations of white-colored films on the surfaces of aquatic macrophytes located within the hydrothermal flow of vents. It was hypothesized that the white films were the product of microbial growth, particularly sulfur-oxidizing bacteria. An investigation of the relevant biological compounds in the vent waters was conducted. Microscopy, non-culture molecular techniques, and phylogenetic analysis were used to assay the bacterial diversity associated with the films. Microscopic analysis of the white films revealed the presence of long filaments (>200 µm) that contained sulfur granules. Filaments with these characteristics were not detected on the normal macrophyte samples. Nucleic acids were extracted from the surface of macrophyte coated with the white film (SB1, SB2) and from the surface of an uncoated macrophyte (SC). 16S ribosomal (rRNA) genes were amplified with the polymerase chain reaction (PCR) and cloned. Amplified ribosomal DNA restriction analysis (ARDRA) was used to examine 100 clones from each library and identify unique phylotypes. S(Chao1) and the Shannon Index, mathematical measures of richness and heterogeneity, were employed to assess the ARDRA pattern diversity of each sample. The SC community contained 50 unique phylotypes, predominantly cyanobacteria and proteobacteria, and was the most heterogeneous. SB1 and SB2 communities were less heterogeneous and dominated by Thiothrix. Dilution to extinction PCR conducted with specific primers indicated that the relative abundance of Thiothrix 16S rRNA gene copies in all three samples were similar. However, reduced sulfur compounds from the vent resulted in a more narrow habitat that supported the sulfur-oxidizing Thiothrix in the white film to the exclusion of cyanobacteria and other proteobacteria found on the normal macrophyte. The majority of 16S rRNA gene sequences obtained in this study displayed similarities ≤98% to any known sequence in public data bases which suggests an abundance of new bacterial species in Sedge Bay.

Age, dose, and time-dependency of plasma and tissue distribution of deltamethrin in immature rats.

The major objective of this project was to characterize the systemic disposition of the pyrethroid, deltamethrin (DLT), in immature rats, with emphasis on the age dependence of target organ (brain) dosimetry. Postnatal day (PND) 10, 21, and 40 male Sprague-Dawley rats received 0.4, 2, or 10 mg DLT/kg by gavage in glycerol formal. Serial plasma, brain, fat, liver, and skeletal muscle samples were collected for up to 510 h and analyzed for DLT and/or 3-phenoxybenzoic acid (PBA) content by high-performance liquid chromatography. Toxicokinetic data from previous experiments of the same design with young adult (PND 90) rats (Kim, K.-B., Anand, S. S., Kim, H. J., White, C. A., and Bruckner, J. V. [2008]. Toxicokinetics and tissue distribution of deltamethrin in adult Sprague-Dawley rats. Toxicol. Sci. 101, 197-205) were used to compare to immature rat data. Plasma and tissue DLT levels were inversely related to age. Preweanlings and weanlings showed markedly elevated brain concentrations and pronounced salivation, tremors, choreoathetosis, and eventual fatalities. Plasma DLT levels did not reliably reflect brain levels over time. Plasma:brain ratios were time and dose dependent, but apparently not age dependent. Brain levels were better correlated with the magnitude of salivation and tremors than plasma levels. Hepatic intrinsic clearance of DLT progressively increased during maturation, as did the hepatic extraction ratio. Thus, limited capacity to metabolically inactivate DLT appeared primarily responsible for the inordinately high target organ doses and acute neurotoxicity in pups and weanling rats. Hepatic blood flow was not rate limiting in any age group. Limited DLT hydrolysis was manifest in vivo in the pups by relatively low plasma PBA levels. Elevated exposure of the immature brain to a pyrethroid may prove to be of consequence for long-term, as well as short-term neurotoxicity.