Impact of pesticide coexposure: an experimental study with binary mixtures of lambda-cyhalothrin (LCT) and captan and its impact on the toxicokinetics of LCT biomarkers of exposure
This study aimed at gaining more insights into the impact of pesticide coexposure on the toxicokinetics of biomarkers of exposure. This was done by conducting an in vivo experimental case-study with binary mixtures of lambda-cyhalothrin (LCT) and captan and by assessing its impact on the kinetic profiles of LCT biomarkers of exposure. Groups of male Sprague–Dawley rats were exposed orally by gavage to LCT alone (2.5 or 12.5 mg/kg bw) or to a binary mixture of LCT and captan (2.5/2.5 or 2.5/12.5 or 12.5/12.5 mg/kg bw). In order to establish the temporal profiles of the main metabolites of LCT, serial blood samples were taken, and excreta (urine and feces) were collected at predetermined intervals up to 48 h post-dosing. Major LCT metabolites were quantified in these matrices: 3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic (CFMP), 3-phenoxybenzoic acid (3-PBA), 4-hydroxy-3-phenoxybenzoic acid (4-OH3PBA). There was no clear effect of coexposure at the low LCT dose on the kinetics of CFMP and 3-PBA metabolites, based on the combined assessment of temporal profiles of these metabolites in plasma, urine and feces; however, plasma levels of 3-PBA were diminished in the coexposed high-dose groups. A significant effect of coexposure on the urinary excretion of 4-OH3PBA was also observed while fecal excretion was not affected. The temporal profiles of metabolites in plasma and in excreta were further influenced by the LCT dose. In addition, the study revealed kinetic differences between metabolites with a faster elimination of 3-PBA and 4-OH3BPA compared to CFMP. These results suggest that the pyrethroid metabolites CFMP and 3-PBA, mostly measured in biomonitoring studies, remain useful as biomarkers of exposure in mixtures, when pesticide exposure levels are below the reference values. However, the trend of coexposure effect observed in the benzyl metabolite pathway (in particular 4-OH3BPA) prompts further investigation.
Keywords: Toxicokinetics; Biomarkers; Coexposure; Pyrethroids; Lambda-cyhalothrin; Captan
Introduction
Insecticides belonging to the pyrethroid family are widely used worldwide because of their broad spectrum of action (Katsuda [21]; Matsuo [29]; Perkins et al. [32]). The most largely used pyrethroids include permethrin, cypermethrin, deltamethrin, lambda-cyhalothrin (LCT) (Matsuo [29]). Pyrethroids represent more than a quarter of global insecticide use and are integrated in agricultural, residential and public health programs to control pests (Aznar-Alemany and Eljarrat [6]). Although pyrethroid insecticides are not persistent in the environment, their frequent use contributes to maintain background levels in the human body as shown by measurements of metabolites in urine and blood samples collected from various populations (Barr et al. [7]; Channa et al. [8]; Choi et al. [10]; Li et al. [26]; Valcke et al. [43]; Wielgomas et al. [46]; Wielgomas and Piskunowicz [47]). In addition to this background exposure, agricultural workers are also exposed in the workplace, making their levels of exposure higher than those of the general population (Ratelle et al. [33]). In these environments, workers are repeatedly or simultaneously exposed to several chemical pesticides.
Numerous efforts have been made in recent years to assess and take into account interactions that could exist between pesticides in coexposure situations. Toxicodynamic and metabolic interactions following exposure to mixtures of pesticides containing pyrethroids were previously studied. The type of interaction varied depending on the molecules and the mechanisms of action. Additive type interactions were evidenced in in vivo experiments (i) looking at motor activity in rodents acutely exposed to mixtures of several pyrethroids (Starr et al. [40], [39]; Wolansky et al. [48]) and (ii) assessing biochemical markers of cytotoxicity in serum of subchronically exposed rodents pyrethroids (cypermethrin) in combination with imidazole and benzimidazole fungicides (imazalil, carbendazime) (Dikic et al. [11]). In in vitro experiments, additive interactions on oestrogen/androgen receptor activity and aromatase activity were also documented when exposing human cell lines to cypermethrin pyrethroid in combination with triazine and triazole fungicides as well as an organophosphorus (OP) insecticide (terbuthylazine, bitertanol, propiconazole, malathion) (Kjeldsen et al. [25]). Another in vitro study reported synergistic inhibition of acetylcholinesterase activity following mixed exposure to pyrethroids (deltamethrin and cypermethrin) in combination with OPs (triazophos, malathion, chlorpyrifos) (Arora et al. [5]). An in vivo study in rodents repeatedly exposed to deltamethrin pyrethroid in combination with thiacloprid also showed a synergistic effect on thyroid hormone levels (Sekeroglu et al. [38]). Metabolism studies further showed that CYP 450-active OPs decreased the ability to detoxify pyrethroids due to inhibition of esterase activity, leading to potentiation effects (Hernández et al. [18]).
Coexposure to various pesticides can also have an impact on the toxicokinetics of the parent compounds and their metabolites. An in vivo study in rats has shown that coexposure to OP insecticides in binary mixture had an impact on their rate of absorption and elimination (Timchalk et al. [42]). With regard to coexposure with pyrethroids more specifically, only a limited number of in vivo rodent studies have compared the toxicokinetics of pyrethroid metabolites after exposure to the compound in mixture and alone (Hirosawa et al. [19]; Wielgomas and Krechniak [45]). The latter studies relate to permethrin and cypermethrin exposures and report a decrease in the excretion of urinary pyrethroid metabolites in rats after coexposure with OP insecticides (Hirosawa et al. [19]; Wielgomas and Krechniak [45]).
Some in vitro studies on cell cultures have further shown that coexposure to pesticides can have an impact on the rate of metabolism of substances (Abass and Pelkonen [1]; Joo et al. [20]; Tang et al. [41]). Other in vitro studies have reported that the main enzymes involved in the biotransformation of pyrethroids, in particular CYP450s (CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2, 3A4, 3A5, 4A1, 2C8, 2C9, 2C19), are also implicated in the biotransformation of several other pesticides used concomitantly in agriculture (Joo et al. [20]; Martinez et al. [28]; Scollon et al. [37]; Yang et al. [49]). In particular, pyrethroids and phthalimides such as captan are metabolized by common CYP450 enzymes, namely CYP3A, CYP1A1, CYP1A2, CYP2A1, CYP2B1 in the liver (Paolini et al. [31]). Phthalimide inhibition of these liver enzymes was noted in this latter study.
The objective of the current study was to gain more insights into the potential impact of pesticide coexposure on the toxicokinetics of biomarkers of exposure. This was done by conducting an in vivo experiment assessing the toxicokinetics of LCT biomarkers of exposure in rats exposed to binary mixtures of LCT and captan compared to LCT alone. LCT was chosen as a key pyrethroid largely used in agriculture and captan because it is used abundantly and concomitantly on crops. The focus was placed on the metabolites of LCT used as biomarkers of exposure in biomonitoring studies, 3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic (CFMP), 3-phenoxybenzoic acid (3-PBA) and 4-hydroxy-3-phenoxybenzoic acid (4-OH3PBA) (Anadon et al. [2]; Khemiri et al. [22], [23]; Schettgen et al. [36]). The chosen LCT doses were based on the No-Observed Adverse Effect Level (NOAEL) of 2.5 mg/kg bw/day and Lowest-Observed Adverse Effect Level (LOAEL) of 12.5 mg/kg bw/day reported by the EPA (2004b) and established from a two-year chronic feeding/carcinogenicity study in rats (Alpk/AP strain) exposed to cyhalothrin and an observed decrease in body weight and food consumption. In an acute neurotoxicity study in Wistar rats orally exposed to LCT at doses of 2.5, 10 or 35 mg/kg bw, the NOAEL for LCT was reported to be the same as that of cyhalothrin, based on clinical signs of neurotoxicity at highest doses and increased breathing rate at the intermediate dose (EFSA [13]). To prevent adverse effects that may be related to acute and chronic ingestion of pesticides in the general population, acute and chronic reference doses (RfDs) of 0.0025 and 0.001 mg/kg bw/day, respectively, were established for LCT based on a chronic neurotoxicity study in dogs exposed orally (NOEL 0.25 and 0.1 mg/kg bw, respectively, divided by an uncertainty factor of 100) (EPA 2004b). These NOEL were not retained in our study given they are based on a dog study rather than a rat study. A RfD has also been derived for captan fungicide. It was established at 0.1 mg/kg bw/day in a three-generation reproduction study in rats based on a NOAEL of 12.5 mg/kg bw to which a safety factor of 100 was applied (EPA [15]).
Materials and methods
Chemicals and reagents
Lambda-cyhalothrin (98.7% purity) and captan (99.2% purity) were purchased from Sigma Aldrich (Saint Louis, USA). Reference standards of 3-PBA, 4-OH3BPA and the internal standards of 13C2 1D trans-DCCA and 13C6-3-PBA (97–98% purity) were obtained from Cambridge Laboratories Inc. (Andover, MA, USA); reference standard of CFMP (> 95% purity) was acquired from ArkPharm. MS grade methanol (MeOH) was purchased from Honeywell. Glacial HPLC grade acetic acid, ethyl acetate and sodium acetate were obtained from Fisher Scientific (Ottawa, ON, Canada). β-Glucuronidase/arylsulfatase (100,000 Fishman U/mL and 800,000 Roy U/mL from Helix Pomatia) was obtained from Roche Diagnostics (Laval, Quebec, Canada).
Animal acclimation, food and housing
Male Sprague–Dawley rats (10 weeks old, 275 ± 50 g each) were purchased from Charles River Canada (St-Constant, Quebec, Canada). Before the experiment, animals were kept in plastic cages in groups of two to three rats and had free access to food (Teklad Global Diets® #2018 from Envigo, Canada) and tap water ad libitum. Prior to dosing, rats were acclimatized to the metabolic cages designed for separate urine from feces. The individual acclimatization of the rats in metabolic cages was done over three days in a gradual manner, i.e. for 1, 2 and 4 h per day on days -3, -2 and -1 before the experiment, respectively. Twelve hours before the experiment, rats were individually housed in stainless steel metabolic cages designed to separate urine from feces, and they were supplied with drinking water containing glucose (D-Glucose: 40 g/L) to induce a physiologic polyuria allowing for frequent urine collections.
Immediately after exposure, rats were put back in their respective metabolic cage for the duration of the experiment. They had continued access to water with glucose for the first 12 h post-exposure and then to tap water for the rest of the experiment. In addition, rats had access to food ad libitum throughout the experiment period. Lighting was maintained on a 12-h light–dark cycle; the ambient temperature was maintained at 22 ± 3 °C and humidity values ranged from 40 to 70%. The experiment was carried out in accordance with OECD Guideline 417. The protocols were approved by the Ethics Committee for Animal Experimentation of the University of Montreal (CDEA approval #17-044).
Throughout the experimental period, animals were observed at least once a day for clinical signs of treatment-related toxicity. The body weight (bw) of each animal, the mass of food and the volume of water consumed were monitored and recorded daily.
Animal treatment
Thirty Sprague–Dawley rats were randomly divided into six groups of five rats each (n = 5 rats per group). Two groups of rats were exposed orally (by gavage) to a single dose of LCT alone (2.5 or 12.5 mg/kg bw) (corresponding to groups G1 and G3, respectively). These doses are equivalent to the no-observed adverse effect level (NOAEL) and the lowest observed adverse effect level (LOAEL) for LCT, respectively (EPA 2004b). Three other groups were each exposed by gavage to a binary mixture of LCT and captan prepared as follows: [NOAELLCT + equivalent mass dose of captan]: 2.5 mg/kg bw + 2.5 mg/kg bw; [NOAELLCT + NOAELcaptan]: 2.5 mg/kg bw + 12.5 mg/kg bw; [LOAELLCT + NOAELcaptan]: 12.5 mg/kg bw + 12.5 mg/kg bw (corresponding to G2, G4 and G5, respectively). The pesticides were dissolved in biological olive oil (Irresistible brand). Ten milliliters of solution was administered per kilogram of body weight for all doses. A control group of five rats was exposed only to the vehicle used for administration. Prior to this experiment, a pilot study was conducted to verify that the lowest dose of LCT administered to rats was enough to allow quantification of metabolites.
To prepare LCT dosing solutions, appropriate mass of LCT was weighed and premixed in olive oil. For the mixture of LCT and captan solution, each pesticide was weighed separately, mixed together in the same flask and dissolved in a single volume of olive oil. All dose solutions were sonicated thrice successively over periods of 10 min until dissolution. The solutions were also vortexed during the preparation and just before preparing the syringes for gavage. All dose solutions were prepared the day before administration and were stored in a dark container at room temperature until usage.
Biological sampling
Blood sampling and isolation of plasma
Blood samples of approximately 400 μL per sampling were collected by the saphenous vein 20 h prior to exposure and sequentially at 0.5, 1, 2, 4, 8, 12, 24, 30 h after exposure. At 48 h postexposure, rats were anesthetized with CO2, then killed by cardiac puncture and whole blood was withdrawn. Blood samples were collected in microtainers or tubes containing K2-EDTA to prevent coagulation. Samples were kept on ice until the plasma was isolated. To separate plasma from red blood cells, blood samples were centrifuged for 15 min at 1500 g, 4 °C. The plasma supernatant were transferred into clearly identified glass tubes serving for extraction of the metabolites and stored at − 20 °C until analysis. Remaining plasma was pooled and then aliquots were prepared to serve as positive controls.
Collection of urine and feces
Urine and fecal samples were collected from each rat at predetermined intervals over a 48 h period, i.e. 0–3, 3–6, 6–9, 9–12, 12–24, 24–30 and 30–48 h after treatment. Control samples were collected during the 12 h before exposure. Once collected, urine volumes were measured using graduated cylinders, and aliquots were prepared in polypropylene Sarstedt tubes; feces were weighed and transferred in glass tubes. Samples were frozen at − 20 °C until analysis.
Tissue sampling
At the time of killing, main organs were excised, including brain, liver, kidneys and gastrointestinal tract (GI). Tissues (except GI) were rinsed with physiological saline solution (to remove blood), blotted dry before being weighed, transferred in vials and stored at − 20 °C until analysis. Control rats were also killed at the same time as the treated rats.
Sample treatment and analysis
Before extraction, all samples were thawed. All matrices were subjected to an enzymatic hydrolysis to obtain the sum of free and conjugated metabolites of LCT. Hydrolysis was followed by solid-phase extraction (SPE) or liquid–liquid extraction before analysis by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-MS-Q-ToF).
Plasma samples (an exact measurement of 150 or 200 μL depending on available volumes and treated directly in the preservation tubes) were spiked with 200 pmol of internal standard mix (13C2 1D-trans-DCCA and 13C6-3-PBA). Enzymatic hydrolysis was carried out by adding 200 μL of sodium acetate buffer (0.1 M, pH 5.0), 4 μL of β-glucuronidase/arylsulfatase enzyme (100,000 Fishman U/mL and 800,000 Roy U/mL from Helix pomatia) and leaving samples for 16 h in a shaking bath at 37 °C. Analytes were then extracted twice with 3 mL of ethyl acetate saturated in water after centrifugation for 20 min at 1500 g, 4 °C. The organic extracts were evaporated under a gentle nitrogen flow, in a water bath at 40 °C. Residues were dissolved in 250 μL of MeOH (100%). Samples were transferred to Eppendorf microtubes, centrifuged for 60 s at 604 g and transferred to HPLC vials for UHPLC-MS-Q-ToF analysis.
Urine samples (1 mL) were spiked with 200 pmol of internal standard mix (13C2 1D-trans-DCCA and 13C6-3-PBA). A volume of 1 mL of sodium acetate buffer (0.1 M, pH 5.0) and 20 μL of β-glucuronidase/arylsulfatase were added to allow hydrolysis for 16 h in a shaking bath at 37 °C. Samples were subjected to solid phase extraction (SPE) with SEP-PAK C18 cartridges (Waters, Milford, MA, USA). Cartridges were conditioned with 4 mL of MeOH and 8 mL of water; samples were loaded on the cartridges, which were then cleaned with 8 mL of water; analytes were eluted from the cartridges with 8 mL of methanol into 10 mL glass tubes. The organic extracts were evaporated until dryness under a gentle nitrogen flow in a water bath at 40 °C. Residues were dissolved in 1 mL of MeOH. Samples were centrifuged 60 s at 604 g to remove remaining solid residues. The methanolic extract was then transferred into HPLC vials for analysis by UHPLC-MS-Q-ToF.
Tissue and fecal samples were individually homogenized using a polytron after adding sodium acetate buffer solution (0.1 M pH 5.0) so as to have a concentration of 500 mg liver, 250 mg kidney, 400 mg brain, 500 mg GI and 250 mg feces in 4 mL. The samples were then spiked with 200 pmol of internal standard mix (13C2 1D-trans-DCCA and 13C6-3-PBA). Liver samples were heated in a water bath at 90 °C for 5 min and then cooled at room temperature. Enzymatic hydrolysis was performed by adding 20 μL of β-glucuronidase/arylsulfatase and shaking in a water bath for 16 h, at 37 °C. The analytes were then extracted with ethyl acetate as described for plasma. After evaporation of the organic extracts, the residues were resuspended in 1 mL of MeOH.
UHPLC-MS-Q-ToF analysis of CFMP, 4-OH3PBA and 3-PBA was performed exactly with the conditions described in Khemiri et al. ([22]). The analytical limit of detection (LOD) was 0.6 and 1.2 pmol/mL of methanolic plasma extract for CFMP and 3-PBA metabolites, respectively. The LODs were 3.9, 0.7 and 2.3 pmol/mL of methanolic urinary extract for CFMP, 3-PBA and 4-OH3PBA, respectively. Depending on the tissue, the LODs varied between 0.4 and 2 pmol/mL of methanolic tissue extract for CFMP between 0.5 and 3 pmol/mL for 3-PBA and 4-OH3PBA.
Data analysis
The time courses of CFMP, 3-PBA and 4-OH3BPA in plasma, urine and feces were reported as a molar percentage of the administered dose. From plasma concentration (C)—time profiles after oral exposure, toxicokinetic parameters were calculated and include the discrete version of the area under the concentration–time curve (AUC), the area under the first moment of concentration–time curve (AUMC), the mean residence time (MRT), the apparent global elimination rate (kelim) constant and elimination half-life (t1/2), with the values for the terminal elimination phase (Gibaldi and Perrier [16]; Hayes [17]). Comparison of toxicokinetic parameter values between groups (G1, G2, G3, G4, G5) was performed by analysis of variance (ANOVA) followed by parametric Bonferroni post tests for comparison between two groups. A value of p < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001).
Results
Effect of coexposure on the time courses of lambda-cyhalothrin metabolites in plasma
Figure 1a and b respectively depict the temporal profiles of CFMP and 3-PBA in the plasma of rats over a 48-h period following ingestion of 2.5 or 12.5 mg LCT/kg bw alone or as a binary mixture with 2.5 or 12.5 mg captan/kg bw. A significant increase in concentrations compared to controls, for which values were below the LOD, was observed in all cases. The plasma profile of CFMP and 3-PBA in rats exposed to the low dose of 2.5 mg LCT/kg bw alone (G1) was similar to that observed in rats exposed to the binary mixtures of this low LCT dose in combination with both the low and high doses of 2.5 and 12.5 mg captan/kg bw (G2 and G4, respectively); this suggests the absence of interaction at this NOAEL dose of LCT (Fig. 1). However, for the high dose groups (12.5 mg/kg bw of LCT alone or in combination with this equivalent mass dose of captan), plasma levels of 3-PBA at time < 24 h were higher in rats exposed to LCT alone (G3) compared to those exposed to the binary mixture (G5); this suggests a possible effect of coexposure on the benzyl metabolite pathway at that dose. More evidently, Fig. 1 shows an apparent effect of the LCT dose (2.5 versus 12.5 mg/kg bw) on the temporal profiles of CFMP and 3-PBA ([G1, G2, G4] vs [G3, G5]).
Graph: Fig. 1 Effect of lambda-cyhalothrin (LCT) coexposure with captan on the time courses of CFMP (a) and 3-PBA (b) in the plasma of rats (expressed as a molar percentage of the administered dose) following oral exposure. Filled circle: G1 = administration of LCT alone (2.5 mg/kg bw); filled square: G2 = coadministration of LCT + captan (2.5 mg/kg bw each); grey triangle: G3 = administration of LCT alone (12.5 mg/kg bw); × : G4 = coadministration of LCT + captan (2.5 mg/kg bw and 12.5 mg/kg bw respectively); grey diamond: G5 = coadministration of LCT + captan (12.5 mg/kg bw each). Symbols represent mean values and bars are standard deviations
The basic toxicokinetic parameters calculated from the plasma profiles of CFMP and 3-PBA for the groups of rats exposed to LCT alone at the low dose (G1 of 2.5 mg LCT/kg bw) or in binary mixture with captan (at 2.5 or 12.5 mg captan/kg bw; G2 and G4, respectively) confirmed the absence of coexposure effect at these doses; the various mean parameter values were not significantly different between groups (G1 vs G2 vs G4) (Table 1). For the groups receiving the high dose of 12.5 mg LCT/kg bw alone (G3) or in combination with the equivalent massic dose of captan (G5), it was not possible to determine the appropriate toxicokinetic parameters and clearly observe elimination, because some animals showed clinical signs of toxicity. These include feeding cessation, diarrhea, jumping in cage, tremor and mobility impairment. Therefore, these groups of animals were killed at 24 or 30 h after exposure to avoid animal suffering.
Comparison of the kinetic time courses of CFMP and 3-PBA in plasma
Regardless of the exposure group (G1, Fig. 2a; G2, Fig. 2b; G4, Fig. 2c), the comparison of the kinetic time courses of CFMP and 3-PBA in plasma also shows a difference in the kinetics of the two metabolites, with a faster elimination of 3-PBA compared to CFMP. Table 1 highlights that the maximum residence time (MRT) and apparent global plasma elimination half-life (t1/2 derived from kelim) was close to two times higher for CFMP compared to 3-PBA, confirming a longer residence time of CFMP in plasma. 4-OH3PBA levels in plasma were not detectable in blood with the developed method.
Graph: Fig. 2 Comparison of the kinetic time courses of CFMP (filled triangle) and 3-PBA (filled circle) in plasma (expressed as pmol/mL/kg bw) of rats following oral administration of LCT alone at a dose of 2.5 mg/kg bw (a) or in combination with captan at a dose of 2.5 mg/kg bw (b) or 12.5 mg/kg bw (c). Symbols represent mean values and bars are standard deviations
Effect of captan coexposure on the urinary and fecal excretion of lambda-cyhalothrin metaboli...
Figure 3 and Table 2 show that there was no clear effect of coexposure with captan (at 2.5 and 12.5 mg/kg bw) on the total percentage of dose recovered as CFMP and 3-PBA in urine over the 0–24 or 0–48 h post-treatment (i.e. G1 vs G2 vs G4 or G3 vs G5). The total percentage of dose excreted as 3-PBA in 0–24 h and 0–48 h urine collections after administration of the binary mixture of 2.5 mg LCT/kg bw LCT and 2.5 mg captan/kg bw (G2) was significantly higher than corresponding values observed in the groups of rats exposed to 2.5 mg LCT/kg bw alone (G1). However, this does not clearly indicate a coexposure effect given that urinary excretion values in rats exposed to the low LCT dose alone (G1) were not significantly different from those observed in rats exposed to the binary mixture of 2.5 mg LCT/kg bw and 12.5 mg captan/kg bw (G4). This is further supported by the lack of significant difference in urinary excretion values between rats exposed to the high LCT dose of 12.5 mg/kw bw alone (G3) and to the binary mixture of 12.5 mg LCT/kg bw and 12.5 mg captan/kg bw (G5). There was also no clear effect of the LCT dose (2.5 versus 12.5 mg/kg bw) on the total percentage of CFMP and 3-PBA recovered in urine.
Graph: Fig. 3 Effect of captan coexposure on the cumulative excretion time courses of lambda-cyhalothrin biomarkers of exposure, CFMP (a), 3-PBA (b) and 4-OH3PBA (c), in rat urine (expressed as a molar percentage of the administered dose) following oral administration. Filled circle: G1 = administration of LCT alone (2.5 mg/kg bw); filled square: G2 = coadministration of LCT + captan (2.5 mg/kg bw each); grey triangle: G3 = administration of LCT alone (12.5 mg/kg bw); × : G4 = coadministration of LCT + captan (2.5 mg/kg bw and 12.5 mg/kg bw, respectively); grey diamond: G5 = coadministration of LCT + captan (12.5 mg/kg bw ach). Symbols represent mean values and bars are standard deviations
Toxicokinetic parameters (mean ± SD) calculated from concentration-time profiles of CFMP and 3-PBA in rats orally exposed to lambda-cyhalothrin alone at a dose of 2.5 mg/kg bw (G1) or as a binary mixture with captan at a dose of 2.5 mg/kg bw or 12.5 mg/kg bw (G2 and G4, respectively)
Parameters | | Exposure group |
|---|
G1 | G2 | G4 |
|---|
AUC (pmol*h/mL/kg bw) (mean ± SD) | AUCCFMP | 31,521 ± 8001 | 39,623 ± 7207 | 38,976 ± 8496 |
AUC3PBA | 57,706 ± 14,103 | 62,623 ± 12,530 | 49,779 ± 3352 |
AUMC (pmol*h2/mL/kg bw) (mean ± SD) | AUMCCFMP | 682,069 ± 185,665 | 940,364 ± 269,169 | 946,770 ± 303,468 |
AUMC3PBA | 680,147 ± 180,193 | 706,438 ± 128,529 | 623,723 ± 67,225 |
MRT (h) (mean ± SD) | MRTCFMP | 21.6 ± 1.2 | 23.5 ± 3.3 | 24.1 ± 3.6 |
MRT3PBA | 11.8 ± 0.6 | 11.3 ± 0.7 | 12.5 ± 0.8 |
Overall apparent plasma kelim (h−1)a (mean ± SD) | kelim_CFMP | 0.046 ± 0.003 | 0.04 ± 0.01 | 0.04 ± 0.01 |
kelim_3PBA | 0.085 ± 0.004 | 0.09 ± 0.01 | 0.08 ± 0.01 |
Overall apparent plasma elimination half-life (h) (mean ± SD) a | t1/2 CFMP | 15.0 ± 0.8 | 16.3 ± 2.3 | 16.7 ± 2.5 |
t1/2 3PBA | 8.1 ± 0.4 | 7.9 ± 0.5 | 8.7 ± 0.6 |
Apparent terminal elimination kβ (h−1) (mean ± SD) | kβ CFMP | 0.08 ± 0.01 | 0.06 ± 0.02 | 0.06 ± 0.02 |
kβ 3PBA | 0.09 ± 0.02 | 0.09 ± 0.01 | 0.09 ± 0.01 |
Apparent terminal elimination half-life (h) (mean ± SD) | t1/2β CFMP | 9.2 ± 1.4 | 12.1 ± 5.1 | 12.0 ± 4.4 |
t1/2β 3PBA | 7.9 ± 1.4 | 7.8 ± 0.9 | 7.4 ± 0.7 |
ANOVA group comparison not significant (G1 vs G2 vs G4) aAn overall elimination rate and half-life were calculated although it is apparent that elimination is biphasic
Conversely, there was a significant difference in the total percentage of dose recovered as 4-OH3BPA in urine over the 0–24 or 0–48 h post-treatment between the different exposure groups (i.e. G1 vs G2 vs G4 or G3 vs G5) with higher values in coexposure groups than groups exposed to LCT alone (G1 vs G4 and G3 vs G5 in 0–24 h urine samples; G1 vs G2 and G1 vs G4 in 0–48 h urine samples) (Table 2). There was also a significant effect of the LCT dose (2.5 versus 12.5 mg/kg bw) on the total percentage of 4-OH3PBA recovered in urine ([G1, G2, G4] vs [G3, G5] except for G1 vs G5 and G2 vs G5).
Cumulative excretion of CFMP, 3-PBA and 4-OH3PBA in urine and feces expressed as percentage of administered lambda-cyhalothrin dose
Matrix | Time interval post-dosing (h) | Metabolite | Cumulative excretion (mean ± SD % dose) |
|---|
Exposure group |
|---|
G1 | G2 | G3 | G4 | G5 |
|---|
Urine | 0–24 | CFMP* | 4.02 ± 3.15e | 1.08 ± 0.55 | 0.93 ± 0.98 | 3.35 ± 1.10 | 0.37 ± 0.34a |
3-PBA*** | 2.13 ± 0.51b | 3.62 ± 0.42ace | 1.74 ± 0.65b | 2.61 ± 0.16 | 1.71 ± 0.56b |
4-OH3PBA*** | 15.2 ± 0.39 cd | 18.9 ± 2.22 cd | 5.97 ± 0.87abde | 37.6 ± 4.14abce | 15.3 ± 3.77 cd |
0–48 | CFMPNS | 8.1 ± 5.91 | 2.68 ± 1.37 | | 5.62 ± 1.02 | |
3-PBA** | 2.29 ± 0.53b | 3.86 ± 0.5ad | | 2.85 ± 0.11b | |
4-OH3PBA*** | 18.3 ± 1.38bd | 23.7 ± 3.01ad | | 46.1 ± 3.33ab | |
Feces | 0–24 | CFMP* | 0.63 ± 0.24 | 0.58 ± 0.48 | 0.15 ± 0.11 | 0.67 ± 0.55 | 0.064 ± 0.05 |
3-PBA* | 0.08 ± 0.03 | 0.097 ± 0.05 | 0.04 ± 0.02 | 0.09 ± 0.05 | 0.03 ± 0.03 |
4-OH3PBANS | 0.32 ± 0.10 | 0.38 ± 0.18 | 0.18 ± 0.13 | 0.25 ± 0.15 | 0.12 ± 0.13 |
0–48 | CFMPNS | 2.26 ± 0.96 | 1.91 ± 1.20 | | 2.17 ± 0.95 | |
3-PBANS | 0.12 ± 0.06 | 0.15 ± 0.07 | | 0.14 ± 0.17 | |
4-OH3PBANS | 0.54 ± 0.22 | 0.58 ± 0.20 | | 0.47 ± 0.18 | |
G1 = LCT alone (2.5 mg/kg bw); G2 = coadministration of LCT + captan (2.5 mg/kg bw each); G3 = administration of LCT alone (12.5 mg/kg bw); G4 = coadministration of LCT + captan (2.5 mg/kg bw and 12.5 mg/kg bw. respectively); G5 = coadministration of LCT + captan (12.5 mg/kg bw each) *p < 0.05, **p < 0.01, ***p < 0.001 for ANOVA group comparison (G1 vs G2 vs G3 vs G4 vs G5); NS = non-significant with p > 0.05 a,b,c,d,e Mean with different exponent (letters) are statistically different with Bonferroni post-hoc test (p < 0.05) aGroup mean are significantly different from G1 bGroup mean are significantly different from G2 cGroup mean are significantly different from G3 dGroup mean are significantly different from G4 eGroup mean are significantly different from G5
Levels of CFMP, 3-PBA and 4-OH3PBA remaining in tissues 48 h after oral administration of lambda-cyhalothrin alone or as a binary mixture with captan (expressed as a percentage of administered dose)
Tissue | Metabolite | Metabolite levels (mean % dose ± SD)a |
|---|
G1 | G2 | G3b | G4 | G5b |
|---|
48 h | 48 h | 30 h | 48 h | 30 h |
|---|
Brain | CFMP | ND | ND | 0.0003 ± 0.0001 | ND | 0.0002 ± 0.0001 |
3-PBA | ND | ND | 0.0003 ± 0.0002 | ND | 0.0002 ± 0.0001 |
4-OH3PBA | 0.00007 ± 0.00001 | 0.00007 ± 0.00001 | 0.001 ± 0.0001 | 0.00008 ± 0.00001 | 0.0003 ± 0.0002 |
GI | CFMP | 2.6 ± 1.3 | 2.6 ± 0.64 | 4.1 ± 1.0 | 2.7 ± 1.4 | 3.2 ± 2.1 |
3-PBA | 0.002 ± 0.001 | 0.002 ± 0.001 | 0.12 ± 0.02 | 0.007 ± 0.006 | 0.10 ± 0.07 |
4-OH3PBA | 0.03 ± 0.01 | 0.03 ± 0.02 | 0.87 ± 0.55 | 0.05 ± 0.02 | 0.65 ± 0.25 |
Kidneys | CFMP | 0.006 ± 0.0006 | 0.008 ± 0.004 | 0.02 ± 0.005 | 0.007 ± 0.004 | 0.01 ± 0.003 |
3-PBA | 0.0008 ± 0.0001 | 0.001 ± 0.0001 | 0.007 ± 0.004 | 0.0009 ± 0.0004 | 0.004 ± 0.003 |
4-OH3PBA | 0.001 ± 0.001 | 0.001 ± 0.001 | 0.02 ± 0.01 | 0.002 ± 0.001 | 0.03 ± 0.02 |
Liver | CFMP | 0.13 ± 0.05 | 0.19 ± 0.10 | 0.27 ± 0.10 | 0.12 ± 0.07 | 0.17 ± 0.05 |
3-PBA | 0.003 ± 0.001 | 0.002 ± 0.001 | 0.04 ± 0.02 | 0.004 ± 0.001 | 0.02 ± 0.01 |
4-OH3PBA | 0.002 ± 0.001 | 0.002 ± 0.001 | 0.03 ± 0.01 | 0.003 ± 0.002 | 0.04 ± 0.03 |
G1 = LCT alone (2.5 mg/kg bw); G2 = coadministration of LCT + captan (2.5 mg/kg bw each); G3 = administration of LCT alone (12.5 mg/kg bw); G4 = coadministration of LCT + captan (2.5 mg/kg bw and 12.5 mg/kg bw, respectively); G5 = coadministration of LCT + captan (12.5 mg/kg bw each) ND Values were below the LOD aValues were too close to the LOD to allow statistical comparison bValues were calculated with n < 5 animals because some rats were killed at 24 h due to observed signs of suffering
Figure 4 and Table 2 show that, when comparing exposure groups, there was no significant effect of coexposure with captan (at 2.5 and 12.5 mg/kg bw) on the total percentage of dose recovered as CFMP, 3-PBA or 4-OH3PBA in feces over the 0–24 or 0–48 h (i.e. G1 vs G2 vs G4 or G3 vs G5). However, there was a non-significant lower percentage of dose recovered as CFMP, 3-PBA and 4-OH3BPA in feces at the higher dose compared to the lower dose (G1 of 2.5 mg LCT/kg bw vs G3 of 12.5 mg LCT/kg bw; G1 of 2.5 mg LCT/kg bw vs G5 of 12.5 mg LCT/kg bw + 12.5 mg captan/kg bw).
Graph: Fig. 4 Effect of captan coexposure on the cumulative excretion time courses of lambda-cyhalothrin biomarkers of exposure, CFMP (a), 3-PBA (b) and 4-OH3PBA (c), in rat feces (expressed as a molar percentage of the administered dose/h/kg bw) following oral exposure. Filled circle: G1 = administration of LCT alone (2.5 mg/kg bw); filled square: G2 = coadministration of LCT + captan (2.5 mg/kg bw each); grey triangle: G3 = administration of LCT alone (12.5 mg/kg bw); × : G4 = coadministration of LCT + captan (2.5 mg/kg bw and 12.5 mg/kg bw, respectively); grey diamond: G5 = coadministration of LCT + captan (12.5 mg/kg bw each). Symbols represent mean values and bars are standard deviations
Similar to plasma results, the urinary and fecal excretion (Figs. 3 and 4 and Table 2) also highlighted differences in the elimination rate of CFMP compared to 3-PBA and 4-OH3BPA. The time courses of CFMP in urine and feces clearly show that elimination was incomplete 48 h after exposure (for groups G1, G2 and G4, which were not sacrificed earlier). Conversely, excretion of 3-PBA and 4-OH3PBA appeared almost complete at that time period. Combined urinary and fecal results further indicate that excreted CFMP and 3-PBA only represented a small percentage of the administered dose with urinary levels being higher than fecal levels for both metabolites (at the group level, up to 8% on average for CFMP in urine and 2% for CFMP in feces; up to 5.6% on average for 3-PBA in urine and 0.15% for 3-PBA in feces) and that 4-OH3PBA was a major metabolite excreted in the urine of rats (representing up to 46% of dose on average in urine and 0.5% of dose in feces).
Tissue residues of lambda-cyhalothrin metabolites
The residual amounts of CFMP, 3-PBA and 4-OH3PBA remaining in tissues of rats 48 h (30 h for the high-dose groups) after oral administration of LCT alone or as a binary mixture with captan are presented in Table 3, and show no marked differences between groups (although values were too close to the limit of detection to allow statistical comparison between groups). For all dose groups, CFMP, 3-PBA and 4-OH3PBA levels in tissues were in the following descending order: gastrointestinal tract (GI) > liver > kidneys > brain. The slower elimination of CFMP as compared to 3-PBA was also evident from the assessment of GI levels at 48 h post-dosing (for rats sacrificed at 48 h (G1, G2, G4), up to 2.6% of dose on average was found as CFMP versus 0.007% as 3-PBA).
Discussion
Effect of coexposure on the time courses of the biomarkers of exposure
This study assessed the effect of pesticide coexposure on the toxicokinetics of LCT biomarkers of exposure, using an experimental in vivo case-study with binary mixtures of LCT and captan. The a priori hypothesis was that captan could interfere with CYP450 metabolism pathway of pyrethroids or excretion mechanisms (Paolini et al. [31]; Wielgomas and Krechniak [45]). Our study showed that when the dose of LCT tested corresponded to the NOAEL, the time profiles of the metabolites CFMP and 3-PBA in plasma were not affected by coadministration of captan. Our data are in line with those of a recent study that assessed the toxicokinetics of 17 pesticides largely measured in biomonitoring studies and their major metabolites in the plasma of female Long-Evans rats following a single oral dose of the complex mixture (Chata et al. [9]). In the latter study, eight families of pesticides were measured, including anilines, carbamates, carboxamides, organochlorines, organophosphates, oxadiazines, phenylpyrazoles and pyrethroids (LCT, permethrin, cypermethrin); captan was not included in the mixture. Metabolites assessed were CFMP and 3-PBA. The average terminal elimination half-life of CFMP estimated in plasma by these authors and the MRT were similar to those obtained in the current study (respective mean t1/2β of 11.0 versus 9.2–12.1 h and MRT of 19.0 versus 21.6–24.1 h). Although cypermethrin and permethrin were present in the mixture in addition to LCT in the study of Chata et al. ([9]), both of which form 3-PBA metabolites, the mean terminal elimination half-life of 3-BPA in plasma and the MRT were also similar to those reported in our study, both in the groups of rats exposed to LCT alone or as a mixture with captan (respective mean t1/2β of 7.0 versus 7.4–7.9 h and MRT of 10.0 versus 11.3–12.5 h). This suggests that our results may be valid for low-dose complex mixtures. Chata et al. ([9]) also concluded that metabolic enzymes were not saturated at their 0.4 mg/kg bw dose of the mixture.
Nonetheless, at the high LOAEL dose of LCT of 12.5 mg/kg bw and high dose of captan in our study, corresponding to its NOAEL of 12.5 mg/kw bw, a possible effect of coexposure on the benzyl metabolite pathway leading to 3-PBA formation could not be excluded. More specifically, plasma levels of 3-PBA at time < 24 h were higher in rats exposed to the high dose of LCT alone (G3) compared to those exposed to the binary mixture of LCT and captan (G5). However, there was no clear effect of coexposure with captan (at 2.5 and 12.5 mg/kg bw) on the total percentage of dose recovered as CFMP or 3-PBA in urine or feces over the 0- to 24- or 0- to 48-h period post-dosing. On the other hand, a significant effect of coexposure on 4-OH3BPA excretion in urine, but not in feces, was observed at both the low and high doses. A potential effect of coexposure was reported in the available animal studies on permethrin and cypermethrin pyrethroids at high doses in binary mixture with the organophosphorus insecticides dichlorvos and chlorpyrifos (Hirosawa et al. [19]; Wielgomas and Krechniak [45]). In the study of Hirosawa et al. ([19]), male Wistar rats pretreated intraperitonealy with the organophosphorus insecticide dichlorvos, at a low or high dose (0.3 or 1.5 mg/kg bw), were then intravenously injected with a single high dose of the pyrethroid cis-permethrin (20 mg/kg bw). The time course of 3-PBA in the urine over a 48-h period in rats pretreated with the high-dose of dichlorvos showed a significantly lower excretion compared to the non-preteated control group (Hirosawa et al. [19]). In the study of Wielgomas and Krechniak ([45]), rats were repeatedly exposed by gavage, 7 days/week for 28 days, either to 10 mg/kg bw of the organophosphorus pesticide chlorpyrifos or to the pyrethroid α-cypermethrin, or to 5 mg/kg bw of each of these compounds as a mixture. The authors reported a 30% reduction in the excretion of the metabolite 4-OH3BPA in the group of coexposed rats (exposed to the pesticide mixture) compared to the group exposed only to α-cypermethrin (Wielgomas and Krechniak [45]). In our study, the effect of coexposure on 4-OH3BPA in urine and plasma results at the high LCT and captan doses prompts further assessment of the potential impact of coexposure on the benzyl metabolite pathway.
In humans, the only controlled data available on the impact of coexposure on the kinetics of pyrethroids and their metabolites concerns a study in volunteers exposed orally to deltamethrin alone at the Acceptable daily intake (ADI) level of 0.01 mg/kg bw or in mixture with the organophosphorus insecticide chlorpyrifos-methyl at an equivalent massic dose (Sams and Jones [35]). From the temporal profiles of 3-(2,2-dibromovinyl)-2,2-dimethyl-(1-cyclopropane)carboxylic acid (DVBA) and 3-PBA metabolites of deltamethrin in urine, the authors reported that there was no statistically significant difference in the apparent excretion half-life and cumulative excretion in urine during the 0- to 24-h period following mixed exposure as compared to administration of deltamethrin alone (on average 7.1 and 10.4 h, respectively, for exposure with deltamethrin alone and as a mixture). This apparent elimination half-life was determined from the attrition slope of urinary levels; its value thus depends on several physiological processes, including metabolism, storage and transfer of blood to urine and feces. Overall, similar to our rat results on the benzyl pathway, but with a different coexposure scenario (pyrethroid/organophosphorus coexposure) from the one studied in the current work, published high dose studies in animals indicate an effect of coexposure (Hirosawa et al. [19]; Wielgomas and Krechniak [45]) while the low dose in humans did not concur to reveal such mixture effect (Sams and Jones [35]).
Comparison of the kinetic time courses of metabolites in plasma
For all exposure scenarios, the current study further showed a difference in the kinetic time courses of CFMP and 3-PBA, with a slower elimination of CFMP from the body compared to 3-PBA. This was confirmed by the longer residence time (MRT) of CFMP in plasma compared to 3-PBA and the incomplete urinary and fecal excretion of CFMP 48 h after exposure (lack of asymptote) contrary to 3-PBA. Upon repeated exposure, CFMP is thus more likely to accumulate in the body; it appears to be the case when comparing CFMP and 3-PBA levels in the plasma on days 7, 30, 45 and 60 in Wistar rats subchronically exposed to a daily oral dose of 6.2 and 31.1 mg LCT /kg bw (Aouey et al. [4]). In the latter study, a dose-dependent increase in levels of LCT metabolites in plasma was also observed following repeated dosing in rats while there were little variations in levels from week to week for a given dose, suggesting that steady-state equilibrium was rapidly reached within the first week of exposure for both metabolites. CFMP was also found in higher concentrations than 3-PBA in liver of the rats subchronically exposed to a daily oral dose of 6.2 and 31.1 mg LCT/kg bw (Aouey et al. [3]). However, given that Wistar rats were exposed in the latter studies (Aouey et al. [3], [4]) while Sprague–Dawley rats were used in the current work, differences in metabolite formation and kinetics between strains of rats cannot be excluded (Kishida et al. [24]; Saito et al. [34]).
On the other hand, in volunteers exposed to LCT by the oral route, Khemiri et al. ([22]) showed similar time courses for CFMP and 3-PBA in plasma, with respective average peak values at 3.1 and 4.0 h post-dosing and mean elimination half-lives of about 5.3 and 6.4 h. Urinary rate time courses were also similar to the plasma concentration–time curves. This highlights species differences in the kinetics of LCT and its metabolites. Moreover, in the assessed volunteers of the study of Khemiri et al. ([22]), on average 21% of LCT dose were excreted as CFMP in urine in the 84-h period post-treatment as compared to 30% as 3-PBA. In the rats of the current study, cumulative excretion of CFMP and 3-PBA in urine respectively amounted to up to 8 and 5.6% on average and corresponding values in feces were 2% and 0.15%; 4-OH3PBA in urine represented a much higher percent of the dose (on average 18 to 48% in the groups exposed to the low LCT dose alone or in mixture with captan) while its fecal excretion was limited (0.5%).
Our results are in line with the unpublished toxicokinetic studies reviewed in the European Commission (EC) report (2014), and conducted by Syngenta according to the OECD guidelines 417 in rats. In a toxicokinetic study in male and female Han Wistar rats exposed orally to a single dose of 1 or 12.5 mg/kg bw of cyclopropyl- or phenoxy- 14C-labeled LCT, oral absorption of labeled LCT was estimated between 7 and 23%. Labeled LCT was recovered mainly in feces (on average 93–95% by 4-days postdosing of 1 and 12.5 mg/kg bw of cyclopropyl-14C-LCT; on average 83–90% by 4-days postdosing of 1 and 12.5 mg/kg bw of phenoxy-14C-LCT) and mostly in the unchanged parent compound form. Urinary levels of 14C-equivalents represented on average 0.8 to 1.5% and 5.8 to 9.7% of the cyclopropyl-14C and phenoxy-14C LCT dose, respectively. In the EC report, it is further indicated that the fraction of LCT dose absorbed in the body was completely metabolized and eliminated mainly via the kidney in the tested animals, that metabolites were excreted mainly as conjugates in urine and bile of rats, and that 4-OH3PBA was the main excreted metabolite (up to 9.3% of dose found in urine and bile). They further reported that there were differences in the kinetics between animals and humans, although the main metabolism steps appear the same (cleavage of the ester bond of LCT and biotransformation products). In rats, 3-PBA was found mainly conjugated to sulfates (EC 2014) while it is considered to be mainly conjugated to glucuronides in humans (Khemiri et al. [22]; Marsh et al. [27]). Oral absorption also appeared much higher in humans based on CFMP excretion in urine (Khemiri et al. [22]; Marsh et al. [27]) when compared to results of this study and rats and those reported in EC (2014).
Effect of dose on the time courses of metabolites
In our study, although there was no clear effect of coexposure on the kinetics of CFMP and 3-PBA in plasma and excreta (urine and feces), the plasma time courses of CFMP and 3-PBA observed revealed an effect of the LCT dose. This was most evident for 3-PBA with a higher retention in plasma at the LOAEL dose of 12.5 mg LCT /kg bw compared to the NOAEL dose of 2.5 mg/kg bw. While there was no clear effect of the dose on the cumulative percentage of CFMP and 3-PBA recovered in urine, a non-significantly lower cumulative percentage of dose was recovered as CFMP and 3-PBA in feces. A significant effect of the LCT dose (2.5 versus 12.5 mg/kg bw) on the total percentage of 4-OH3PBA recovered in urine was also observed and there was a non-significant trend on the fecal excretion.
Similar to our findings, in the unpublished study conducted by Syngenta and reported in EC (2014), after administration of phenoxy-14C LCT in Wistar Han rats, somewhat lower average cumulative urinary excretion values of 14C equivalents were found both in males and females at the high dose of 12.5 mg/kg bw (5.8 and 6.7%, respectively) compared to the low dose of 1 mg/kg bw (9.7 and 8.1%, respectively). This was also apparent in bile with cumulative excretion values representing on average 3.3% in both male and female rats at the high dose compared to 6.3 and 12% of dose in males and females, respectively at the low dose. Such difference in urinary and biliary excretion was not found after administration of cyclopropyl-14C LCT.
Other studies on pyrethroid metabolites failed to show an effect of dose on their toxicokinetic parameters. In a study looking at the effect of dose on the kinetics of transfluthrin metabolites, Yoshida ([50]) reported that values of the fraction of metabolites excreted in urine, the mean residence time in body (MRT) and half-life period of the urinary excretion rate (t1/2) were dose-independent for each metabolite. In studies on the kinetics of deltamethrin in Sprague–Dawley rats, Mortuza et al. ([30]) also showed that plasma toxicokinetic parameters of deltamethrin (MRT, elimination half-life t1/2 from plasma, clearance Cl, oral absorption rate constant kabs, bioavailability F) were independent of the dose, and maximum concentration Cmax and area under the plasma concentration time-course (AUC) values were directly proportional to the dose.
Furthermore, in the current study, the LOAEL dose of LCT administered alone or as a mixture with captan caused toxicity in some of the rats (tremor, impairment of locomotion), such that they had to be euthanatized. The toxicity thus appeared to be related to LCT administration. Similarly, in an experiment in male Sprague–Dawley rats acutely exposed by gavage to 10 or 20 mg/kg bw of LCT (dissolved in corn oil), Weiner et al. ([44]) observed signs of toxicities 4 h after exposure (i.e. the time of peak effect) at both doses. The authors reported a disappearance of the signs of toxicity in the surviving subjects 24 h after treatment.
Conclusion
Our study showed that exposure to LCT alone at the NOAEL dose or as a binary mixture with captan, which are metabolized by common enzymes (including CYP450), did not alter the toxicokinetics of the metabolites CFMP and 3-PBA commonly used in biomonitoring studies to assess exposure to LCT. These findings confirm the usefulness of these pyrethroid metabolite measurements to assess absorbed doses in biomonitoring studies in individuals exposed to low levels of pesticide mixtures. However, the trend of coexposure effect observed for 3-PBA at the LOAEL dose of LCT and for 4-OH3BPA prompts further investigation. The impact of coexposure on the toxicokinetics of LCT biomarkers of exposure is nonetheless limited to binary mixtures rather than complex mixtures and high doses that may not be relevant to humans; there is also evident biological variability that may render interpretation more difficult. The method for the measurement of 4-OH3BPA in plasma should also be optimized in order to further document the effect of coexposure on the benzyl pathway leading to the formation of this metabolite. Future perspectives include verifying coexposure effect in potentially highly exposed individuals, such as workers.
Funding
This study was funded by the Chair in Toxicological Risk Assessment and Management of the University of Montreal and by the Institut de recherche en santé publique of the University of Montreal (now renamed Centre de recherche en santé publique) funded by the Fonds de recherche en santé du Québec (FRQS).
Compliance with ethical standards
Conflict of interest
Authors declare no conflicts of interest.
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By Yélian Marc Bossou; Jonathan Côté; Marc Mantha; Sami Haddad; Sophie Achard and Michèle Bouchard
Reported by Author; Author; Author; Author; Author; Author
