110s6.part2

Protein Binding of Isofluorophate in Vivo after Coexposure
to Multiple Chemicals
John S. Vogel, Garrett A. Keating II, and Bruce A. Buchholz
University of California, Lawrence Livermore National Laboratory, Livermore, California, USA 14C/mouse). A dynamic range of 1,000 in a Full toxicologic profiles of chemical mixtures, including dose–response extrapolations to realistic
tracing experiment thus needs no more than exposures, is a prohibitive analytical problem, even for a restricted class of chemicals. We present
2 nCi or 75 Bq tracer/animal. The Nuclear an approach to probing in vivo interactions of pesticide mixtures at relevant low doses using a
Regulatory Commission has long ruled that a monitor compound to report the response of biochemical pathways shared by mixture compo-
yearly nonradioactive disposal of up to 1 µCi nents. We use accelerator mass spectrometry (AMS) to quantify [14C]-diisopropylfluorophosphate
as a tracer at attomole levels with 1–5% precision after coexposures to parathion (PTN), perme-
concentrations <50 nCi/g is acceptable as thrin (PER), and pyridostigmine bromide separately and in conjunction. Pyridostigmine shows an
nonradioactive waste (7). With a total of overall protective effect against tracer binding in plasma, red blood cells, muscle, and brain that is
<2 nCi/30-g mouse, this disposal limit is well not explained as competitive protein binding. PTN and PER induce a significant 25–30%
above the levels in AMS tracing experiments.
increase in the amount of tracer reaching the brain with or without pyridostigmine. The sensitiv-
The typical AMS sample contains 1 mg car- ity of AMS for isotope-labeled tracer compounds can be used to probe the physiologic responses of
specific biochemical pathways to multiple compound exposures. Key words: accelerator mass spec-
brain tissue, or the red blood cells (RBCs) trometry, chemical mixture, diisopropylfluorophosphate, esterase, organophosphate, parathion,
from 15 µL whole blood. These small sample permethrin, pyrethroid, radioisotope. Environ Health Perspect 110(suppl 6):1031–1036 (2002).
sizes are readily available from mice. DFP is http://ehpnet1.niehs.nih.gov/docs/2002/suppl-6/1031-1036vogel/abstract.html
available at high specific activity (~2 14C/mol-ecule), making it a particularly sensitive probeof delivered protein inhibition. DFP retains and a simulant of offensive nerve agents that this 14C label when bound to enzymes and is inhibits target proteins through covalent surroundings, diet, or medications. Most of attachment. This study included parathion these exposures occur at very low doses for irreversibly to serine hydrolases in the brain, which individual chemical toxicity is not most commonly used insecticides of the OP and pyrethroid classes, respectively. An addi- and in skeletal muscle, including membrane could result. Environmental toxins occur at tional quaternary cholinesterase inhibitor, esterases. It also binds to soluble plasma pyridostigmine bromide (PYB), was added to hydrolases, including coagulation factors, mass) for which studies of high-dose mecha- the group to represent natural cholinergics or carboxylesterase, and butyrylcholinesterase nisms may no longer apply. Full toxicologic protective pharmaceutical intake. The chem- (BChE), and to cytosolic serine proteases, profiles of all chemical mixtures, including but is hydrolyzed to the nonbinding diiso- dose–response extrapolations to realistic exposures believed related to Gulf War syn- propylphosphoric acid (DPA) by circulating exposures, is a prohibitive analytical prob- drome (4) but is also relevant to domestic and hepatic A-esterases (8,9). Although DFP lem, even for a restricted class of chemicals exposures of insecticides and pediculocides.
is frequently used as an enzyme inhibitor to such as pesticide residues on food. We pre- identify the targets of serine-based hydrolysis sent an approach to probing in vivo interac- at levels of 0.1 µmol/kg, even for children (5).
(10), the complement and relative affinities tions of toxin mixtures at relevant low doses, These trace concentrations are insufficient for quantifiable inhibition of enzymes through in vivo is unknown. Two primary protein response of a biochemical pathway shared by activity assays, the standard technique for targets in homogenized rat brain in vitro are mixture components. This approach has the OP-reactive sites, along with six or more potential for revealing unexpected interac- exposures of < 0.01 µmol/kg/day to maintain minor targets (11); similar numbers of both tions arising from physiochemical effects undisturbed enzymatic activities. Highly sen- sitive quantitation of the monitor compound This article is part of the monograph Application ofTechnology to Chemical Mixture Research. modifications of tissue binding using com- environmental concentrations or less.
Address correspondence to J.S. Vogel, Center for Accelerator Mass Spectrometry, Lawrence Livermore pesticides. The toxic mechanism of OPs is isotope-labeled compounds whose labels have National Laboratory, 7000 East Ave., L-397, primarily disruption of the cholinergic path- Livermore, CA 94551 USA. Telephone: (925) 423- way in nerve transmissions. Cholinesterase radioisotopes. Isotope decay detection is inef- 4232. Fax: (925) 423-7884. E-mail: [email protected] inhibition is seen in high-dose exposures of ficient, however, so we used accelerator mass This research was supported by National Institute of Environmental Health Sciences ES 09690, most OPs, with a wide variance in effective spectrometry (AMS) to quantify a 14C iso- National Center for Research Resources RR13461, toxicity (1,2). Serine hydrolases are found in topic label on tracer compounds at attomole and University of California-Campus Laboratory levels with 1–5% precision (6). This sensitiv- Collaboration 95-103. One reviewer made signifi- immunity and respiration, and these proteins ity quantitates even tracer doses in highly cant contributions in conceptual clarification. This are also classic OP targets with potential fractionated tissue samples from small animal work was performed in part under the auspices of toxic consequences (3). We chose diiso- models such as mice. A 30-g mouse contains the U.S. Department of Energy by University ofCalifornia Lawrence Livermore National Laboratory about 1.4 becquerels (Bq) (37 pCi) of natural compound because it is an anticholinesterase 14C, and AMS easily quantifies tracer 14C at Received 18 December 2001; accepted 4 October pharmaceutical, a binder to serine hydrolases, 5% above natural (75 milliBq or 2 pCi tracer Environmental Health Perspectives • VOLUME 110 | SUPPLEMENT 6 | DECEMBER 2002 membrane and cytosol proteins are found in Materials and Methods
The blood was centrifuged at 1,000 rpm for homogenized chicken brain in vitro (12).
Chemicals. [14C]-DFP (specific activity = 160
Samples were either processed immediately likely targets or extent of DFP binding that England Nuclear Life Science Products, Inc.
for AMS analysis or stored at –70°C.
Chemical dosing. Chemicals for
metabolism in living animals at low doses.
dose–response and chemical mixture studies The hydrophobic DFP will primarily report were presented in food to reduce stress to the levels of dissolution within lipids, binding to animals. Mice were placed on a fast/feed regi- cell membrane proteins, or to plasma-soluble men that removed food in the evening, pre- proteins. Our procedure attempted to pre- PER were purchased from Chem Service (PS- vent postdissection binding to cytosol pro- 95 and PS-758; West Chester, PA, USA) and morning containing the particular chemical assayed > 98.5% purity. PYB and unlabeled dose, and resumed availability of dry food follow-up of this study will use remaining ad libitum until the next evening. The moist tissues to identify DFP-binding proteins in D0879). [14C]-DFP was diluted in unlabeled food consisted of a 1/1.6 mixture of Purina DFP to adjust specific isotope activities to desired levels. Purina 5K92 Pico-Vac Rodent Nutrition International)/distilled water pre- pared each morning. This food/water consis- bound to proteins at very low doses, and any tency prevented the mice from handling the unbound compound is rapidly cleared (13).
Interanimal and even intratissue variations in highest grade readily available from commer- food were transferred by spatula into the bulb hydrolase concentrations are expected to be of a plastic disposable transfer pipette that much greater than the trace concentration of Test animals. CD2/F1 male mice were
was cut with scissors so food retained in the bulb was accessible to the mice. Chemicals enzyme activity assays respond to these ani- MA, USA) at 6–8 weeks of age and quaran- were added to the food in 10–40 µL veg- mal and tissue variations, which mask physio- tined for 5 days for acclimatization and to etable oil or ethanol diluent vehicles. The logically induced changes at tracer levels. Our modified pipettes containing the food were reporter fluctuations are instead expected to Association for Assessment and Accreditation wedged between the bars of the wire cage top reflect physiologic or physiochemical changes of Laboratory Animal Care–approved animal brought about by coexposed chemicals that care facility for experiments. Protocols were modulate the amount of reporter reaching a tissue or binding to enzymes. Stress can affect pipettes were removed from the cage. More cerebral-vascular transmission (14), requiring than 80%, >95% in most cases, of the moist experimental design that either quantifies or minimizes stress effects that might mask those Dose–response study. Five mice per dose
group were acclimated to the fast/feed regi- designed our experiment to eliminate stress housed individually in plexiglass containers men for 5 days prior to dosing. The moist by avoiding handling of the mice throughout with wire cage tops and lined with shaved provided ad libitum, except as noted below, 10–40 µL oil or alcohol vehicle containing during a 12-hr light/dark cycle at 20°C.
the radiolabel. Doses of 14C-labeled PER, numerous biologic pathways, as they possess hormonal activities (15), affect enzymatic regulation (16), induce membrane perme- dures to minimize stress-induced differences abilities (17), and produce cytotoxicity were calculated from body mass at euthanasia in vitro (18), among other physiologic effects Pharmacokinetic study. Mice received
and percentage of consumed carrier food.
not directly related to their esterase interac- intraperitoneal (ip) DFP doses of 100 ng/kg tions. Chronic low-level exposures are impli- unlabeled equivalents to maintain the optimal cated in cognitive dysfunctions (19), but pled for tissue kinetics at 1, 12, 24, 48, and tissue content of 14C for accurate AMS quan- 168 hr. DFP was dissolved in hexane/poly- titation (approximately 2 nCi/mouse, pro- shown no detrimental cognitive effects at ethylene glycol and added to peanut oil for chronic daily doses ranging up to 500 µg/kg.
50–100 times the natural 14C). Control mice However, direct acetylcholinesterase (AChE) sacrificed by CO2 asphyxiation at appropri- ate times. Blood (0.25–0.5 mL) was imme- with an equal volume of vehicle. Dry food doses (20). Thus, enzyme-binding com- diately obtained by cardiac puncture with and water were available ad libitum until euthanasia at 48 hr postdosing. Blood and tis- probes of how chemical mixtures of pesti- transferred to plastic tubes containing 2–3 sues were harvested as described above. Two cides might affect physiology and chemistry drops of EDTA, mixed, and stored on ice.
mice per PTN dose group were transferred to but should have little effect on mouse health clean animal bedding immediately after dos- and behavior. The goal of our work was to and stored on ice: a piece of the right femur ing. Fecal pellets were sieved from the cage quantify any changes in tissue exposures of muscle, liver, spleen, and brain. The brain bedding of these mice to determine bioavail- was cut in half sagitally, the left hemisphere ability by quantifying unabsorbed [14C]-PTN preexposure to other anthropogenic esters at in the pellets. Pellets were homogenized and the very low doses obtained from household plastic tube, and both tubes stored on ice.
VOLUME 110 | SUPPLEMENT 6 | DECEMBER 2002 • Environmental Health Perspectives Chemical Mixtures • Exposure to multiple chemicals Hillsdale, NJ, USA) for liquid scintillation counting. Recovery efficiency of the oxidizer was measured at 99.2% using spiked controls.
exposure effects were analyzed for significance elimination is seen in the first 12 hr. The 1-hr Chemical mixture study. Moist food in
(p ≤ 0.05) using unpaired, 2-tailed t-test with data for RBC and brain imply inefficiencies presentation pipettes was supplemented with InStat software (Graphpad Software Inc., San of removing plasma from these samples at 10 blank solvent, all with and without 50 µg/kg with the standard error in the median. Very higher reaction rate with plasma protein, PYB through a 5-day fast/feed cycle. Four broad data distributions were prevalent in the including BChE (350/µM-hr), than with the homogenized, and in short-time kinetic data moist food presentation. Dry food and water with rapid changes in tissue concentrations.
were available ad libitum until euthanasia at than plasma-bound levels and very low brain labeling in the early exposure. The long-term plasma clearance time (48 hr) matches that Tissue preparation. The left hemisphere
confirming near-normal distributions.
found by Martin (13), but the brain binding of each brain was placed in a 2.0-mL plastic relative to plasma is a factor of 10 lower than tube with 1.0 mL water prior to violent agi- Results and Discussion
The bioavailability of PTN delivered in the higher dose. The brain clearance is more than (Biospec Products, Bartlesville, OK, USA) moist food ranged from 98.7 to 99.7%, based a factor of 2 slower than found in that high- for 10 sec to disrupt the cells and solubilize on the measured 14C content of the feces col- sents 16 pmol to a 30-g mouse whose blood volume is 1.3–1.6 mL (21), for a maximum sequential centrifugations at 10,000×g in (<20%) retention of the PTN isotope label at blood concentration of 11 nM. This repre- 0.5 mL water for 8 min each, with immedi- 48 hr, indicating that the majority of the label sents just 0.001% of the Michaelis-Menten ate supernatant removal to avoid cell mem- was hydrolyzed and excreted as p-nitrophenol branes refloating from the pellet. Sequential in urine between dosing and euthanasia. Any blood and tissue (22), allowing linear model- washes indicated more than 99% of the sol- urine contamination of the recovered fecal ing that assumes the concentrations of meta- pellets would only decrease the quantified bolic enzymes remain essentially unchanged removed from the tissue after three rinses.
bioavailability, an effect that must be minimal Lipid membrane material was not separated clearance. The measurements and model rep- that the chemical delivery in food was efficient resent the regeneration and elimination of the all bound isotope was assumed to represent and avoided the confounding physical factor enzyme-substrate pair, as neither the metabo- of stress on the study animals from handling lized free DPA nor the recycled protein com- ponents are able to further label proteins.
by centrifugation and removal of the plasma.
plasma, RBC, and brain are shown in Figure 1 plasma, and rinsed brain containing 1–5 mg carbon were packaged for AMS sample prepa- with fitted curves from a simple compartmen- ration. All samples were dried in a vacuum tal model of protein-bound DFP in Figure 2.
parameters (in units per hour) are shown in Figure 2. A storage and release compartment, introduction into the LLNL AMS ion source.
assumed to be lipid, is required to correctly Statistics. Three to five mice provided
model the retention in plasma binding at 24 data for each experimental condition. All hr. We have seen similar kinetic behavior for other lipophilic pesticides in mice (23), and four mice. Kinetic data were analyzed by a the Martin’s kinetic profiles of DFP show the same delayed peak (13). Peak AChE inhibi- tion occurs 24–48 hr after PTN and chlor- pyrifos exposures (24). Anticholinesterases esterase in vitro (25), casting some doubt on the need for this purely physical delay com- partment. However, the rapid metabolism of physical protection of the fluorine during Figure 2. The compartmental model used to fit the
this delay. The lipid partition coefficient for DFP concentration
measured kinetics of the ip dose of DFP shows the DFP is more than 10 times that for liver, transfer coefficients of the fit plotted in Figure 1.
kidney, and perfused tissue (22), so the Time postdose (hr)
plasma pool: circulating free DFP, the metabolized assumed model of lipid storage delay is plau- phosphoric acid of DFP (DPA), and the compound Figure 1. Temporal behavior of protein-bound
bound to soluble enzymes (EZM). Other compart- represent a delayed chemical interaction.
[14C]-DFP is shown for plasma (circle), RBC (dia- ments include the liver (LIV), RBCs, and brain mond), and brain (square) after a 100-ng/kg ip (BRN). Clearance from bound components to dose. Model concentrations are given for both retention is achieved primarily through pro- excreta (XCR) is assumed rapid compared with bound (dotted lines) and total components of DFP plasma enzyme turnover. Transfer coefficients Environmental Health Perspectives • VOLUME 110 | SUPPLEMENT 6 | DECEMBER 2002 sampling the mixture-exposed animals at that than unrecognized threshold effects of a single Chemical mixture study. Table 1 gives the
median tissue concentrations of [14C]-DFP, lifetime for unbound PER, implying that the dietary intake in Figure 3 showed low survival with and without subchronic PYB, at ppt for 48-hr delay is sufficient in clearing that from first-pass metabolism for DFP and PER, groups of animals unexposed (DFP only) and unbound compound also (26). Enzyme with plasma concentrations only 5–10% the regeneration or cell turnover removing the averaged body dose. Metabolism and clear- PER together. Control data for animal han- tracer label from both RBC and brain is mod- dling, tissue isolation, and measurement were eled at 7.5 days mean lifetime. The modeled reduce the plasma concentration of intact obtained from animals receiving equivalent treatments with moist food containing only similar to measured lifetimes of other plasma 0.1% of the exposure concentration at 48 hr.
vehicle solvents. These control tissues all con- proteins, such as albumin (27).
Plasma partitioning to RBC was similar for tained within 2% of the expected natural 14C Dose–response study. The dose responses
lite p-nitrophenol, showed preferential uptake, natural 14C was subtracted from all tissue assurance that differences detected in multi- perhaps lipophilic, to RBC over remaining in measurements before converting the remain- chemical exposures were mixture effects rather plasma. PER partitioned into the brain at only ing 14C signal to equivalent [14C]-DFP con- 10% the rate of DFP for equivalent body dose (Figure 3C), despite its high lipophilicity.
represents <0.03 pg DFP equivalents/g tissue Retained PTN label had significantly nonlin- and is incorporated into the uncertainties ear dose response in the plasma and brain, stated in the table and shown in figures. Liver with relatively greater loss of the labeled frac- and spleen tissues were not measured from tion at higher doses. PER showed significant the PYB groups. These highly perfused tissues nonlinearity in plasma, with greater retention retained high levels of bound DFP even after at higher doses. Dose responses were linear 48 hr, up to 10 times those found in plasma.
within measurement and fitting uncertainties concentration of DFP tracer as plasma, RBC, threshold effects were noted near the planned retention tissues arise from sampling and cir- culatory variations but also from the measure- exposures differ markedly from those of the ment procedure. AMS is highly sensitive for ip exposures used in determining kinetics.
low levels of isotope, but instrumental correc- tions and uncertainties increase at the high 79 ± 16% and 53 ± 13%, respectively, of that concentrations found in these tissues.
of plasma concentration, as opposed to the 20 and 5.4% levels observed at 48 hr in the ip binding in tissues, without (Figure 4A) and kinetic data. The extrapolated response for an oral 100-ng/kg dose predicts a brain concen- coexposures did not affect absorption or dis- tration of 0.5 pg/g, a factor of 2 lower than tribution of the reporter DFP, as seen by the the concentration measured 48 hr after the ip constant plasma, muscle, liver, and spleen Tissue concentration (pg/g)
dose. In contrast, the plasma and RBC con- centrations for oral exposure were a factor of consistent changes in DFP binding for all 30 and 10, respectively, lower than those from ip exposure at equivalent extrapolated doses. Digestive hydrolysis of DFP to a non- exposures, respectively. The increases persist binding DPA may be significant in oral expo- sures, greatly reducing the active component available for binding to circulating protein and cells, but that low plasma concentration, square root of the sum of the squares) of the perhaps distributed on lipoproteins, remained nearly as effective in entering the brain and could arise through greater absorption or Table 1. The median values of [14C]-DFP concentrations are expressed in picograms DFP per gram tissue
(± the standard error in the median) for the blood and four tissues from mice coexposed to pesticides and
Dose (µg/kg)
Figure 3. Tissue concentrations of [14C]-PTN, [14C]-
PER, and [14C]-DFP in plasma, RBC, and brain are shown as functions of the orally administered dose corrected for mouse body mass and dose con- sumption. Power fits to the data are shown. The only significantly nonlinear data are for PTN residues in plasma and brain and PER in plasma. No threshold effects are found near 1-µg/kg doses.
Measurement uncertainties are present on all data.
VOLUME 110 | SUPPLEMENT 6 | DECEMBER 2002 • Environmental Health Perspectives Chemical Mixtures • Exposure to multiple chemicals C]-DFP concentration (pg/g)14[
concentrations + PYB/– PYB
Brain Muscle Spleen
Figure 5. Percentage change of DFP binding in
Figure 4. Concentrations of [14C]-DFP bound in plasma, RBC, brain, muscle, liver, and spleen due to
plasma, RBC, brain, and muscle due to subchronic preexposure of mice to pesticides at 1-µg/kg doses, without (A) and with (B) preexposure to 50 µg/kg PYB.
coadministration of PYB at 50 µg/kg prior to DFP Uncertainties are propagated standard errors in the medians of groups of four or five mice. Asterisk (*) exposure along with other chemical exposures.
indicates significant differences from the case of no added pesticides.
PYB shows an overall protective effect that is sig-nificant in data for the brain and pesticide-free greater retention. Greater retention implies RBC. Asterisk (*) indicates significant differences from the case of no added pesticides.
influenced by the presence of the other com- changes in [14C]-DFP tissue concentrations pounds. AChE activity in rat brain signifi- Conclusions
cantly increased after 60 mg/kg PER dosed both subchronically for 7 days and acutely show some significant differences consistent tissue-specific effects due to low-dose exposures (28), along with modifying other enzyme- among all pesticide exposures. PYB does not to multiple compounds. The concentrations of easily pass the rodent blood–brain barrier, bound DFP in all tissues reflect the level of sure dose was 0.002% of that level and was even under stressed conditions (31,32), active compound reaching the tissue shortly although one study suggests otherwise (33).
after the oral [14C]-DFP exposure, with possi- hydrolases in the brain. Synergistic effects ble modulation of uptake, distribution, and such as multisite binding that effectively binding due to the preexposed compounds.
whole-body average for PYB) that can cause (29,30). The DFP delivered to the brain is only minor competitive inhibition of pro- significantly change the amount of toxin that teins. Abou-Donia et al. see no significant reaches the brain at concentrations that would change in brain enzyme activities for AChE, not have measurable effects on traditional enzyme activity assays. These pesticides were hundreds nanomolar (28), providing binding acetylcholine (ACh) receptor, and nicotinic provided by oral exposure doses that are com- proteins already in large excess of the DFP ACh receptor at PYB doses 25 times greater mensurate with doses available through normal and minimizing the probability of coincident than those used here, despite significant ingestion of sprayed foods, drinking of surface binding on a given protein. Thus, induction waters, or use of home pesticides. The mecha- of enzymatic production by pesticides should most affected by PYB alone (34). Our nism appears to be an increase in cerebral-vas- cular transmission of DFP that is independent of protective effects from transient binding by might indicate an overall process of circula- extrapolation of those higher-dose studies.
PYB. If the increase in brain DFP concentra- tion or brain absorption nearing saturation, An induction of protective esterase activity tions is due to greater barrier permeability, elsewhere in the body leading to lower DFP absorption processes sensitive to the pesti- plasma concentration is consistent with our obtain greater access to brain tissue during cides in varying degrees. The brain absorp- observation, and we do see a decrease of DFP low-level pesticide exposure. This would rep- resent an increase in neural toxicity that without pesticides. Any such induction of results from alternative mechanisms from the required for the latter interpretation.
enzyme activity was specific to elimination of primary decrease in enzyme activity, a usual Pesticide disturbance of the hypothesized measure of toxic end points. The sensitivity of lipid storage (Figure 2) and reemergence of the DFP preferentially in the lipid-filled by PYB. PYB may possess a protective mech- can be used to probe the physiologic responses brain could contribute to the observed effect, of specific biochemical pathways to multiple but tissue specificity would have to be high competitive enzyme binding possible at these for the brain signal to be so unlike the RBC doses. This PYB dose is only 4% of the pro- or muscle, which show no pesticide-induced phylactic dose used by service people in the REFERENCES AND NOTES
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