The effect of plasma lipids on the pharmacokinetics of chlorpyrifos and the impact on interpretation of blood biomonitoring data.

Lipophilic molecules, like chlorpyrifos (CPF), present a special problem for interpretation of biomonitoring data because both the environmental dose of CPF and the physiological (pregnancy, diet, etc.) or pathological levels of blood lipids will affect the concentrations of CPF measured in blood. The objective of this study was to investigate the distribution of CPF between plasma and tissues when lipid levels are altered in late pregnancy. CPF was sequestered more in the low-density lipid fraction of the blood during the late stages of gestation in the rat and returned to nonpregnant patterns in the dam after birth. Plasma partitioning of CPF increased with increases in plasma lipid levels and the increased partitioning of CPF into plasma lipids resulted in less CPF in other tissue compartments. Gavage dosing with corn oil also increased plasma lipids that led to a moderate increase of CPF partitioning into the plasma. To mechanistically investigate the potential pharmacokinetic effects of blood lipid changes, an existing CPF physiologically based pharmacokinetic/pharmacodynamic model for rats and humans was modified to account for altered lipid-tissue partition coefficients and for major physiological and biochemical changes of pregnancy. The model indicated that plasma CPF levels are expected to be proportional to the well-known changes in plasma lipids during gestation. There is a rapidly growing literature on the relationship of lipid profiles with different disease conditions and on birth outcomes. Increased blood concentrations of lipophilic chemicals like CPF may point to altered lipid status, as well as possibly higher levels of exposure. Thus, proper interpretation of blood biomonitoring data of lipophilic chemicals requires a careful consideration of blood lipids.

Investigations of oxidative stress, antioxidant response, and protein binding in chlorpyrifos exposed rat neuronal PC12 cells.

ABSTRACT Chlorpyrifos (CPF) is a widely used organophosphate insecticide. In addition to its known properties of cholinesterase inhibition, the production of reactive oxygen species (ROS) has been suggested as a possible toxic mechanism. To investigate CPF-generated ROS, rat neuronal PC12 cells were exposed to CPF concentrations of 0 to 5000 mug/mL in Krebs buffered media (KRH), KRH + 4% bovine serum albumin (BSA), and KRH + 25 muM of the antioxidant Trolox for 0 to 5 h. Paraquat served as a positive control for ROS. The fluorescent probe 2,7-dichlorodihydro-fluorescein and the MTS assay were used to measure ROS and cytotoxicity, respectively. Examinations into CPF-albumin binding were also conducted. CPF was not strongly cytotoxic to PC12 cells, causing only mild cytotoxicity at 5000 mug/ml. In KRH media, CPF-generated ROS was observed at 4 and 5 h at 500 and 1000 mug/mL, and at 1 to 5 h at 5000 mug/mL CPF. In KRH + 4% BSA, ROS was seen only at 5 h in 5000 mug/mL CPF. Trolox significantly reduced CPF- and paraquat-induced ROS. Calculated CPF-albumin binding at 1, 10, and 100 mug/mL CPF in 4% BSA was 96%, 75%, and 15%. These data show CPF at >/=500 mug/mL induced ROS in PC12 cells, but the addition of the antioxidant Trolox and 4% BSA dramatically reduced ROS levels.

The effect of route, vehicle, and divided doses on the pharmacokinetics of chlorpyrifos and its metabolite trichloropyridinol in neonatal Sprague-Dawley rats.

There is a paucity of data on neonatal systemic exposure using different dosing paradigms. Male CD (Sprague-Dawley derived) rats at postnatal day (PND) 5 were dosed with chlorpyrifos (CPF, 1 mg/kg) using different routes of exposure, vehicles, and single versus divided doses. Blood concentrations of CPF and its primary metabolite, trichloropyridinol, were measured at multiple times through 24 h. Groups included were single gavage bolus versus divided gavage doses in corn oil (one vs. three times in 24h), single gavage bolus versus divided gavage doses in rat milk, and sc administration in dimethyl sulfoxide (DMSO). These data were compared with lactational exposure of PND 5 pups from dams exposed to CPF in the diet at 5 mg/kg/day for 4 weeks or published data from dams exposed to daily gavage with CPF at 5 mg/kg/day. Maternal blood CPF levels were an order of magnitude lower from dietary exposure than gavage (1.1 vs. 14.8 ng/g), and blood CPF levels in PND 5 pups that nursed dietary-exposed or gavage-exposed dams were below the limit of detection. Single gavage doses of 1 mg/kg CPF in corn oil vehicle in pups resulted in CPF blood levels of 49 ng/g and in milk vehicle about 9 ng/g. Divided doses led to lower peak CPF levels. A bolus dose of 1 mg/kg CPF in DMSO administered sc appeared to have substantially altered pharmacokinetics from orally administered CPF. To be meaningful for risk assessment, neonatal studies require attention to the exposure scenario, since route, vehicle, dose, and frequency of administration result in different systemic exposure to the test chemical and its metabolites.

Subchronic feeding study of DAS-59122-7 maize grain in Sprague-Dawley rats.

59122 is a transgenic maize line containing event DAS-59122-7 that expresses the corn rootworm (CRW) specific pesticidal Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis (Bt) Berliner strain PS149B1 and the phosphinothricin-N-acetyltransferase (PAT) protein from Streptomyces viridochromogenes for tolerance to the herbicidal ingredient glufosinate-ammonium. For the current study, 59122 maize grain, non-transgenic near-isogenic maize grain (091), and a commercially available non-transgenic reference maize grain (33R77) were grown under conditions simulating commercial farming practices. Adult Sprague-Dawley rats (12/sex/group) were fed diets formulated with 35% maize grain from either 59122, 091, or 33R77, or one of two separate lots of commercially available rodent chow prepared with commercially available corn (35%) in accordance with the standards of Purina Mills Labdiet 5002 for approximately 90 days. All diets possessed similar nutritional and contaminant profiles. The transgenic proteins were detected only in diets prepared with 59122 maize grain and were stable over the course of the study. Compared to control groups, no adverse diet-related differences were observed in rats fed diets formulated with 59122 maize grain with respect to body weight/gain, food consumption/efficiency, clinical signs of toxicity, mortality, ophthalmology, neurobehavioral (FOB and motor activity) assessments, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), and pathology (organ weights and gross and microscopic pathology). Results from this study indicate that 59122 maize grain is nutritionally equivalent to and as safe as conventional maize grain.

Mixtures in the real world: the importance of plant self-defense toxicants, mycotoxins, and the human diet.

A perusal of research presented at the Annual Society of Toxicology Meetings, or in nearly any toxicology journal, will show that the overwhelming emphasis of toxicology research is on synthetic chemistries. Because of substantial potency and exposure to natural chemicals, the overwhelming focus on synthetic chemistries cannot lead to a realistic understanding of chemical risk to the general population. Natural chemicals, simply because of their abundance and potency, may be as likely to be a public health concern and to be involved in chemical interactions (natural:natural, natural:pharmaceutical; or natural:synthetic) as are environmental levels of synthetic chemicals. All plants have a mix of natural self-defense chemistries and mycotoxins that, when tested in a manner comparable to synthetic pesticides, cause the entire spectrum of toxic effects. As a further complication, plants also escalate much of their self-defense chemistry when attacked by insects and fungi, and damaged crops often have higher mycotoxins levels. Effective crop protection will typically reduce the plant's levels of self-defense toxicants and mycotoxins, but may add residues of synthetic pesticides or add some other risk variable. In addition, cooking may also alter the food chemistry (e.g., acrylamide). The mixtures toxicologist needs to address the real world mixture of natural and synthetic chemicals. Public policy on crop-food safety cannot be sensibly guided without these data and large voids in our understanding of risks from real-world mixtures cannot be in the public interest.

Dose-effect analyses of occupational chlorpyrifos exposure and peripheral nerve electrophysiology.

We performed nerve conduction studies (NCSs) on 113 chemical workers, many of whom had occupational exposure to the organophosphorus insecticide chlorpyrifos (O,O-diethyl-O-[3,5,6-trichloro-2-pyridyl]-phosphorothioate), to identify dose effects of subclinical neuropathy. In this masked longitudinal study, we estimated historic and interim chlorpyrifos exposures and measured excretion of 3,5,6 trichloro-2-pyridinol (TCP), a chlorpyrifos metabolite. TCP excretion among exposed workers suggested an estimated daily chlorpyrifos exposure averaging about 576-627 microg/day and indicated levels approximately 30% (range 0-250%) of the internal dose received by a typical subject exposed during a working day at the threshold limit value of 200 microg/m3. We modeled NCS results using linear mixed models with repeated measures. Although we found no consistent associations between interim chlorpyrifos exposure and NCS results, we identified several significant associations involving historic chlorpyrifos exposure. Most associations, however, reflected effects at low-exposure levels (< 20 mg/m3 x days) without further effects as exposure increased over a 10-fold range (20-220 mg/m3 x days). This suggested small differences among subjects with low or no chlorpyrifos exposure, rather than a dose-related deterioration among subjects with higher exposures. Two NCS results demonstrating apparent subclinical adverse dose effects showed significant but unexplained interaction with education level. The overall results provide little support for the hypothesis that chronic chlorpyrifos exposures at levels in the range associated with appreciable inhibition of B-esterases produce adverse dose effects on peripheral nerve electrophysiology suggestive of subclinical neuropathy.

Thirteen week feeding study with transgenic maize grain containing event DAS-Ø15Ø7-1 in Sprague-Dawley rats.

Maize line 1507, containing event DAS-Ø15Ø7-1 (1507), is a genetically modified (GM) maize plant that expresses the cry1F gene from Bacillus thuringiensis (Bt) sbsp. aizawai and the phosphinothricin-N-acetyltransferase (pat) gene from Streptomyces viridochromogenes throughout the plant including in the grain expression of the Cry1F protein confers in planta resistance to the European corn borer (ECB; Ostrinia nubilalis Hübner: Crambidae) and other lepidopteran pests. Expression of the PAT protein confers tolerance to the herbicidal active ingredient glufosinate-ammonium. The current study evaluated the nutritional performance of rats fed diets containing 1507 maize grain in a subchronic rodent feeding study. The grains in this study, 1507, its near-isogenic control (33P66), and a non-GM commercial hybrid (33J56) contained similar amounts of proximates, amino acids, minerals, anti-nutrients, and secondary metabolites. The subchronic feeding study compared standard toxicology response variables in rats fed diets containing 1507 maize grain with those in rats fed diets containing non-GM maize grains. All diets were prepared according to the specifications of PMI Nutrition International, LLC Certified Rodent LabDiet 5002 (PMI) 5002). Diets were fed ad libitum to Sprague-Dawley rats for approximately 90 days. In-life response variables included indicators of dietary performance and weekly evaluations for clinical signs of toxicity. No toxicologically significant differences were observed in the nutritional performance variables, clinical and neurobehavioral signs, ophthalmology, clinical pathology (hematology, clinical chemistry, coagulation, and urinalysis), organ weights, and gross and microscopic pathology between any pair of treatment groups. These results demonstrate that 1507 maize grain is as safe and as nutritious as non-GM maize grain.

Differential sensitivity of blood, peripheral, and central cholinesterases in beagle dogs following dietary exposure to chlorpyrifos.

Two studies were performed to find out whether exposure limits that protect brain acetylcholinesterase (AChE) will protect peripheral tissue AChE after exposure to chlorpyrifos (CPF), an organophosphate insecticide. In a methods-development study, male dogs (3/dose) were exposed to 0.0, 0.3, 0.6, or 1.2mg/kg/day CPF in their diets for 4 weeks. Mixed cholinesterase (mChE), AChE, and butyrylcholinesterase (BuChE) activities were measured in plasma, RBC, brain, left atrium and ventricle, diaphragm, quadriceps, and nodose ganglia. Plasma, brain and peripheral tissue BuChE was inhibited at all dose levels. While RBC AChE was inhibited at all doses, brain and peripheral AChE activities were unaffected. In the main study, dogs (4/sex/dose) were exposed to 0.0, 0.5, 1.0, or 2.0mg/kg/day CPF in their diets for six weeks and RBC AChE was significantly inhibited at all doses in both sexes. Diaphragm, quadriceps, and nodose ganglia AChE was unaffected by treatment. Brain AChE was decreased by approximately 6% compared to controls in high-dose groups, and this was considered a threshold effect. Left atrium AChE in high-dose dogs was 25.5% less (males) and 32.1% greater (females) than controls; these differences were attributed to chance. While peripheral tissue and brain AChE were not affected following exposure to 1.0mg/kg/day, RBC AChE was inhibited at all doses. These results show that RBC AChE is more sensitive than brain or peripheral tissue AChE to inhibition by CPF, and that protection of brain AChE would protect peripheral tissue AChE.

Lack of trigeminal nerve toxicity in rats exposed to trichloroethylene vapor for 13 weeks.

Male and female Fischer-344 rats were exposed to 1,1,2-trichloroethylene (TCE) at 250, 800, or 2500 ppm for 6 h/day, 5 days/week, for 13 weeks. Weekly body weights and daily clinical observations were recorded and a functional observational battery (FOB) was performed monthly. Postexposure neurotoxicological evaluations included an electrodiagnostic evaluation of auditory function, the trigeminal nerve, and a comprehensive neuropathological examination. After 8 weeks of exposure, female, but not male, rats exposed to 2500 ppm were slightly more reactive to handling than the controls but not after 13 weeks of exposure. After 13 weeks, female rats exposed to 2500 ppm TCE were slightly more active during the 1-min observation period than the controls. There were no treatment-related differences in grip performance, landing foot splay, or on the trigeminal nerve-evoked potential at any dose. At 2500 ppm TCE, mild frequency-specific hearing deficits were observed, including elevated tone-pip auditory brainstem response thresholds. Focal loss of hair cells in the upper basal turn of the cochlea was observed in 2500 ppm-exposed rats. Except for the cochleas of 2500 ppm-exposed rats, no treatment-related lesions were noted during the neuro-histopathologic examination. The no-observable-adverse-effect level for this study was 800 ppm based on ototoxicity at 2500 ppm.

Lack of cross-reactivity between the Bacillus thuringiensis derived protein Cry1F in maize grain and dust mite Der p7 protein with human sera positive for Der p7-IgE.

Cry1F protein, derived from Bacillus thuringiensis, is effective at controlling lepidopteran pests and a synthetic Cry1F transgene was transferred into maize. For the safety assessment of genetically modified food crops, the allergenic potential of the introduced novel trait(s) is evaluated. Because no single parameter is currently predictive of allergic potential, a 'weight of evidence' approach has been proposed. As part of this assessment, the amino acid (aa) sequence of the Cry1F protein was compared to a database of known allergens using recommended criteria. The Cry1F protein did not show significant similarity or a match of eight contiguous identical aa with any allergen. However, a single six contiguous aa match was identified between Cry1F and the Der p7 protein of the dust mite, Dermatophagoides pteronyssinus. To investigate whether Cry1F was cross-reactive with Der p7, sera from 10 dust mite allergic patients containing Der p 7-specific IgE antibody were used to compare IgE-specific binding. No evidence of cross-reactivity was observed between Cry1F and Der p7. This study provides in vitro IgE sera screening data, that when considered in the context of other bioinformatic data [Hileman R.E., Silvanovich, A., Goodman R.E., Rice E.A., Holleschak G., Astwood J.D., Hefle S.L., 2002. Bioinformatic methods for allergenicity assessment using a comprehensive allergen database. Int. Arch. Allergy Immunol. 128, 280-291; Stadler, M.B., Stadler, B.M., 2003. Allergenicity prediction by protein sequence. FASEB J. 17, 1141-1143.], adds further evidence arguing against the use of a six contiguous identical amino acid search to identify potential cross-reactive allergens. Cry1F is heat labile, rapidly hydrolyzed in an in vitro pepsin resistance assay, not glycosylated and not from an allergenic source. Taken together, these data indicate a lack of allergenic concern for Cry1F.