Abstract
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.
Introduction
Sales of pyrethroid insecticides have increased substantially worldwide due to their effectiveness and restrictions on the use of organophosphates (Williams et al., 2008). A variety of pyrethroids is widely used agriculturally and in households for pest control. Studies of general populations reveal that a large proportion of those surveyed has been exposed to permethrin (PER) and several other pyrethroids (Morgan, 2012; Saillenfait et al., 2015). Children often exhibit higher urinary pyrethroid metabolite levels than adults, apparently due largely to ingestion from hand-to-mouth activities involving contaminated surfaces. PER, commonly sold as a mixture of its 14C-cis-permethrin (CIS) and 14C-trans-permethrin (TRANS) isomers, is the most widely used insecticide in the United States for residential use. PER is also often the treatment of choice for infestation of pets and children with lice and other parasites (Anadón et al., 2009; Frankowski and Bocchini, 2010).
High doses of pyrethroids can be acutely neurotoxic, although their potency differs substantially (Wolansky et al., 2006). Sufficient doses of type I pyrethroids elicit tremors and sensitivity to external stimuli. Type II pyrethroids can produce salivation, hyperexcitability, and choreoathetosis. The parent compounds are the proximate neurotoxic entities. They produce stimulus-dependent depolarization block, primarily by interference with voltage-sensitive sodium channels (Cao et al., 2011; Soderlund, 2012). Disturbance of voltage-sensitive sodium channels may result in residual impairment of the maturing nervous system (Shafer et al., 2005). Several recent epidemiology studies have found an association between long-term pyrethroid exposure and neurobehavioral disorders in children (Richardson et al., 2015; Viel et al., 2015; Wagner-Schuman et al., 2015; van Wendel de Joode et al., 2016). Other researchers failed to find an association between prenatal or postnatal PER exposure and neurodevelopmental problems (Horton et al., 2011; Quirós-Alcala et al., 2014).
It has been recognized for 25 years that immature rodents are more susceptible to high-dose pyrethroid neurotoxicity than adults. PER, a type I pyrethroid, as well as cypermethrin and deltamethrin (DLM), type II pyrethroids, were found to be much more acutely toxic to neonatal than to adult rats (Cantalamessa, 1993; Sheets et al., 1994). Kim et al. (2010) subsequently observed that DLM levels in plasma, brain, and other tissues were inversely proportional to rats’ stage of development. Deficient metabolic detoxification and systemic clearance by liver cytochrome P450s (P450s) and carboxylesterases (CaEs) and plasma CaEs in the immature animals were reported to be a cause of this phenomenon (Anand et al., 2006). Low adipose tissue content in neonatal and preweanling rats was observed to promote enhanced deposition of the highly lipophilic pyrethroids in the central nervous system (CNS) (Amaraneni et al., 2017a). Postnatal day (PND) 15 and 21 rats with plasma DLM levels comparable to adults have recently been observed to exhibit significantly higher brain levels, suggesting that the immature blood-brain barrier (BBB) is relatively permeable to the chemical (Mortuza et al., 2018). An in situ experiment revealed that brain uptake of DLM was inversely related to the age of rats (Amaraneni et al., 2017b). A goal of the current investigation was to determine whether this was also true for PER, the insecticide to which children are most frequently exposed in the United States.
The pharmacokinetics, as well as the acute neurotoxic potency of CIS and TRANS, is quite different. Pilot pharmacokinetics experiments revealed that rats of different ages can tolerate substantially higher single oral doses of TRANS. TRANS is eliminated more rapidly from the blood and brain of rats than CIS (Tornero-Velez et al., 2012; Willemin et al., 2016). CIS is metabolized solely by P450s, whereas TRANS is oxidized by P450s and extensively hydrolyzed by CaEs in rats (Scollon et al., 2009). The ester linkage of the TRANS configuration of PER is quite labile to CaE-catalyzed hydrolysis (Hosokawa, 2008). It is also possible that CIS is more potent than TRANS because CIS penetrates the immature BBB more readily.
Materials and Methods
CIS and TRANS (each of 99.9% radiochemical purity) were provided by Moravic (Brea, CA). Hanks’ balanced salt solution was obtained from Mediatech (Manassas, VA). Human serum albumin was purchased from Golden West Biologicals (Temecula, CA). Ecolite scintillation cocktail was obtained from MP Biochemicals (Solon, OH), whereas glycerol formal (GF) was purchased from Sigma-Aldrich (St. Louis, MO).
Adult males (∼300 g), dams with ∼10 pups, and pregnant female rats (gestation day 17–19) were purchased from Charles River Laboratories (Raleigh, NC). The Sprague–Dawley rats were delivered to and housed at the Association for Assessment and Accreditation of Laboratory Animal Care International–accredited University of Georgia Life Sciences Animal Care Facility. The animals were acclimated to a 12-hour light/dark cycle (light 0700–1900 hours) in a temperature (25°C)- and humidity (40%)-controlled room for at least 1 week prior to use. Tap water and Purina Rat Chow 5001 were provided ad libitum. Adult male rats were housed four per solid–bottom polycarbonate cage. Dams with pups and pregnant females were housed singly in the cages with corncob bedding. The pregnant rats’ delivery date was defined as PND 0 for their pups. Pups were maintained with their mother until PND 15 or 21. Groups of four adults and three to five unsexed pups were used for each experiment according to the research protocol approved by the University of Georgia Animal Care and Use Committee. The study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The initial activity of both 14C-CIS and 14C-TRANS was 61 mCi/mmol. A stock solution of each was prepared by dissolving 6.4 mg in 2 ml GF. Aliquots of stock were used to make working solutions of 10, 100, and 500 µM each isomer in GF. Two hundred microliters of working solution were added to 1800 µl 4% human serum albumin in Hanks’ balanced salt solution to obtain final concentrations of 1, 10, and 50 µM for infusion.
Adult rats were anesthetized by i.m. injection of 0.1 ml/100 g bodyweight of a cocktail consisting of ketamine hydrochloride (100 mg/ml), acepromazine maleate (10 mg/ml), and xylazine (20 mg/ml) (3:2:1, v/v/v). PND 15 and 21 rats received the same dose i.p. The common carotid artery of adults was exposed and cannulated with a 23G needle attached to PE-50 tubing. It was necessary to use a 30G needle affixed to PE-10 tubing to cannulate PND 15 and 21 pups. Each animal’s cardiac ventricles were then cut to stop the flow of blood to the brain before infusion was begun, as well as to allow drainage of the perfusate. Two milliliters (for adults) or 0.5 ml (for pups) saline was then slowly injected into the cannula to flush as much blood as possible from the brain’s vasculature. Dosing solutions were incubated at 37°C in an orbital shaker for 15 minutes prior to injection. A Harvard syringe pump was used to infuse dosing solutions into the left brain of each rat at a rate of 500 µl/min for 4 minutes in adults and 250 µl/min for 2 minutes in pups. After the infusions were completed, 2 (for adults) or 0.5 ml (for pups) saline was slowly perfused through the cannula to flush unabsorbed test compound from the vasculature. In situ brain perfusion is reported to be one of the most sensitive and reliable techniques for estimating CNS uptake of xenobiotics (Bickel, 2005).
The left half of the brain was removed after perfusion and processed for measurement of the two isomers. Portions of brain were homogenized in two volumes of ice-cold distilled water with a Tekmar Tissuemizer. One milliliter of brain homogenate was added to 4 ml Ecolite scintillation cocktail, and each isomer was quantified by liquid scintillation counting with a Beckman Coulter LS 6500 (Pasadena, CA) and normalized for tissue weight.
Data were analyzed and expressed as mean ± S.D. by use of Microsoft Excel (Microsoft, Redmond, WA). The statistical significance of differences between groups was assessed by one-way analysis of variance, followed by Tukey’s multiple comparison test, with a significance level of P < 0.05, using GraphPad Prism 5.01 (GraphPad Software, San Diego, CA).
Results and Discussion
Maturation of the BBB had a pronounced effect on penetration by both CIS and TRANS in situ. Brain uptake of each of the three infused concentrations of CIS and TRANS was significantly greater in the PND 15 than in the PND 21 or PND 90 rats (Table 1). The extent of CNS deposition of 1 µM each isomer, a toxicologically relevant concentration, was significantly different in all three age groups. Brain uptake of 10 and 50 µM CIS and TRANS consistently appeared to be slightly higher in PND 21 weanlings than adults, but the apparent differences were not sufficient to be statistically significant. The magnitude of difference in pyrethroid permeation, as a function of stage of BBB maturation, is included in parentheses in Table 1. Interestingly, uptake of 1 µM each isomer decreases by ∼50% with each stage of maturation.
Relatively little information was found in the literature on changes in BBB permeability to xenobiotics during development of rodents or humans. The BBB of PND 10–11 rats was reported to be more permeable than that of adults to the lipophilic corticosteroid triamcinolone (Arya et al., 2006). Cerebral deposition of inulin progressively decreased from PND 4–26 in rats (Ferguson and Woodbury, 1969). Structural integrity of the BBB was reported to be achieved by PND 21, the time of weaning of rats (Schulze and Firth, 1992). Tightening of endothelial junctions occurred in conjunction with increasing thickness of the basement membrane and envelopment by pericytes. Pericyte, astrocyte, and basement membrane signaling were reported to maintain integrity of the tight cell junctions (Liebner et al., 2011). Pediatric studies involving i.v. drug injection and cerebrospinal fluid (CSF) monitoring have not provided definitive information on the progression of permeability changes in the BBB or chorid plexus of humans. Most investigations have been limited to neonatal and adult subjects. The most definitive data have been obtained from measurements of CSF protein levels and dye penetration. CSF protein levels decrease rapidly during the first 6 months of life (Wong et al., 2000), with the highest levels measured 2–4 weeks after birth (Shah et al., 2011). Misra et al. (1987) reported an inverse relationship between entry of fluorescein into the CSF and age up to 6 months, with the largest drop in CSF fluorescein levels during the first 2 weeks. It might be assumed from the foregoing that reasonably effective BBB function is achieved within 2–4 weeks of parturition in both rodents and humans. The immature rat thus appears to be a reasonable animal model with which to forecast CNS dosimetry in infants and children (Semple et al., 2013).
The magnitude of age-dependent differences in pyrethroid permeation, as a function of the stage of BBB maturation, is included in parentheses in Table 1, which includes measured brain concentrations. BBB permeability varied with the concentration of infused CIS and TRANS in rats in the current investigation. Uptake of 1 µM CIS and TRANS is 5.4- to 5.5-fold greater in the brain of the least mature pups than in the adult brain (Table 1). The disparity in brain levels between pups and adults was less pronounced at the two highest CIS and TRANS concentrations. Saturation of an active influx process with limited capacity in immature rats may be responsible for this phenomenon. A low-affinity influx transporter was described for DLM, CIS, and TRANS uptake by caco-2 cells, as well as DLM uptake by hCMEC/D3 (human brain microvascular endothelial) cells (Zastre et al., 2013; Amaraneni et al., 2016).
The isomer appeared to have little effect on the extent of CNS deposition of CIS and TRANS in any age group in the present study. Very similar levels of the two isomers were measured in the brain of animals infused with 1 µM (Table 1). CIS concentrations consistently appeared to slightly exceed TRANS concentrations for the 10 and 50 µM infusions. The apparent prominence of CIS increased with infused concentration and animal age, although CIS deposition was significantly higher only in adults perfused with 50 µM.
Brain uptake of both CIS and TRANS was linear in all three age groups of rats over the 50-fold range of concentrations (Fig. 1). R2 values for all ages of animals exceeded 0.999. Amaraneni et al. (2017b) also found brain uptake of DLM to be linear. In vitro experiments with CMEC/D3 cells revealed uptake of DLM to be a passive, nonsaturable process (Amaraneni et al., 2016). BBB penetration by xenobiotics has been observed to increase with lipophilicity up to a point, beyond which higher Log P values result in diminishing permeation (Banks, 2009). DLM’s Log P and mol. wt. are 6.1 and 505.2 (ATSDR, 2003). The similarly high Log P (6.5) and molecular mass (391.3) of permethrin satisfy two of the criteria of Lipinski et al. (2001) for poor membrane permeability. Lipid partitioning, coupled with hydrophobic bonding, serves to reduce the flux of large, highly lipophilic chemicals such as pyrethroids through the BBB, virtually trapping them there in membranes (Waterhouse, 2003; Liu et al., 2011). The magnitude and the age dependency of brain uptake of DLM in rats in situ (Amaraneni et al., 2017b) were strikingly similar to CIS and TRANS in the current study. Comparable brain uptake of CIS and TRANS indicates that dissimilar BBB permeation does not contribute to the greater acute neurotoxic potency of CIS in rats. Although the permeability of the BBB to pyrethroids is limited, it appears likely that increased permeability of the immature BBB contributes to the elevated target organ dosimetry manifest in this age group (Kim, et al., 2010; Mortuza, et al., 2018), possibly resulting in enhanced neurologic effects of the insecticides during early development.
Authorship Contributions
Participated in research design: Mortuza, Edwards, White, Cummings, Bruckner.
Conducted experiments: Mortuza, Edwards, Patel.
Performed data analysis: Mortuza, White, Patel, Cummings.
Wrote or contributed to the writing of the manuscript: Mortuza, Bruckner.
Footnotes
- Received October 10, 2018.
- Accepted December 3, 2018.
This work was supported by the Council for the Advancement of Pyrethroid Human Risk Assessment and by the University of Georgia Interdisciplinary Toxicology Program.
Abbreviations
- BBB
- blood-brain barrier
- CaE
- cytochrome P450
- CIS
- 14C-cis-permethrin
- CNS
- central nervous system
- CSF
- cerebrospinal fluid
- P450
- cytochrome P450
- DLM
- deltamethrin
- GF
- glycerol formal
- PER
- permethrin
- PND
- postnatal day
- TRANS
- 14C-trans-permethrin
- Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics