Pharmacological inhibition of CXCR2 chemokine receptors modulates paraquat-induced intoxication in rats

Abstract

Paraquat (PQ) is an agrochemical agent commonly used worldwide, which is allied to potential risks of intoxication. This herbicide induces the formation of reactive oxygen species (ROS) that ends up compromising various organs, particularly the lungs and the brain. This study evaluated the deleterious effects of paraquat on the central nervous system (CNS) and peripherally, with special attempts to assess the putative protective effects of the selective CXCR2 receptor antagonist SB225002 on these parameters. PQ-toxicity was induced in male Wistar rats, in a total dose of 50 mg/kg, and control animals received saline solution at the same schedule of administration. Separate groups of animals were treated with the selective CXCR2 antagonist SB225002 (1 or 3 mg/kg), administered 30 min before each paraquat injection. The major changes found in paraquat-treated animals were: decreased body weight and hypothermia, nociception behavior, impairment of locomotor and gait capabilities, enhanced TNF-α and IL-1β expression in the striatum, and cell migration to the lungs and blood. Some of these parameters were reversed when the antagonist SB225002 was administered, including recovery of physiological parameters, decreased nociception, improvement of gait abnormalities, modulation of striatal TNF-α and IL-1β expression, and decrease of neutrophil migration to the lungs and blood. Taken together, our results demonstrate that damage to the central and peripheral systems elicited by paraquat can be prevented by the pharmacological inhibition of CXCR2 chemokine receptors. The experimental evidence presented herein extends the comprehension on the toxicodynamic aspects of paraquat, and opens new avenues to treat intoxication induced by this herbicide.

Figure 1. Paraquat survival curve and general schedule of treatment used in the study.

(A) Survival curve for cumulative paraquat administration through 28 days, given at different doses [5 (n = 4), 10 (n = 14), 15 (n = 4), and 20 (n = 6) mg/kg], twice a week. (B) The animals received five i.p. applications (at 0, 3, 7, 10, and 14 days) of vehicle/saline (10 ml/kg; group 1), SB225002 (1 and 3 mg/kg; groups 2 and 3)/saline, vehicle/paraquat (10 mg/kg; group 4), or SB225002 (1 and 3 mg/kg)/paraquat (10 mg/kg); groups 5 and 6, respectively. Behavioral tests were performed at days 0, 7, and 14, which corresponded to the basal timepoint, and the 3^rd or the 5^th application of drugs. In some cases, the behavioral measurements were also carried out under basal conditions (prior to any treatment).

Figure 2. Physiological changes elicited by paraquat: effects of SB225002.

(A) Changes in body weight (Δ grams), before the 3^rd and 5^th application; (B) variation of body temperature (Δ °C), measured immediately before treatments; (C) variation of body temperature measured 1 h after each application. Each column represents the mean ± SEM of 4–18 animals per group. ^*p<0.05 and ^**p<0.01 significantly different from control (vehicle/saline) group; ^#p<0.05 significantly different from vehicle/paraquat group (PQ) (ANOVA followed by Bonferroni's post-hoc test).

Figure 3. Behavioral scores of nociception.

Assessment of behavioral scores at 25 min (A) or 55 min (B) after each treatment. Data is plotted as the cumulative scores of nociception over 5 min. Each column represents the mean ± SEM of 4–18 animals per group. ^*p<0.05 and ^**p<0.01 significantly different from control (vehicle/saline) group; ^#p<0.05 and ^##p<0.01 significantly different from vehicle/paraquat group (PQ) (ANOVA followed by Bonferroni's post-hoc test).

Figure 4. Evaluation of paraquat effects in the open-field test.

(A) Number of crossed squares; (B) number of rearings; (C) facial grooming in s; (D) number of fecal boluses. Each column represents the mean ± SEM of 4–18 animals per group. ^*,§p<0.05; ^**,##,§§p<0.01 compared with the basal measurements of the same experimental group (ANOVA followed by Bonferroni's post-hoc test).

Figure 5. Locomotor skills in the beam-walking test.

(A) Time taken to cross the beam in s; (B) number of falls; (C) number of paw slips. Each column represents the mean ± SEM of 4–18 animals per group (ANOVA followed by Bonferroni's post-hoc test was used for analysis of data in Figure 5A).

Figure 6. Symptoms of motor dysfunction.

Representative walking footprint patterns after the 5^th treatment in (A) control (vehicle/saline) group; (B) vehicle/paraquat (PQ)- treated animals; (C) SB225002 (1 mg/kg)/plus PQ group; (D) SB225002 (3 mg/kg)/plus PQ group. The bar graphs depict quantitative analysis of the footprint test regarding the stride length mean (E) and the average step frequency (F). Each column represents mean ± SEM of 9–19 animals per group. ^*p<0.05 and ^**p<0.01 compared with control (vehicle/saline) group (ANOVA followed by Bonferroni's post-hoc test).

Figure 7. RT-qPCR analysis of inflammatory markers.

Relative expression profiles of (A) NF-κB1, (B) TNF-α, (C) IL-1β, and (D) COX-2, assessed in the striatum after the 5^th paraquat treatment. Each column represents the mean ± SEM, 3–4 samples of striatum. ^*p<0.05 compared with control (vehicle/saline) group (ANOVA followed by Bonferroni's post-hoc test).

Figure 8. Hematological analysis and lung neutrophil migration.

Representative pictures showing the blood smear slides in (A) control (vehicle/saline) groups; (B) vehicle/paraquat (PQ)-treated animals; (C) SB225002 (1 mg/kg)/saline group; (D) SB225002 (3 mg/kg)/saline group; (E) SB225002 (1 mg/kg)/plus PQ group; (F) SB225002 (3 mg/kg)/plus PQ group. Effects of paraquat and SB225002 on the total blood cell counts (G) and neutrophil migration as assessed by MPO activity (H). Each column represents the mean ± SEM of 4–20 animals per group in blood cell counts and 3–12 animals per group in MPO activity. ^{**,⊥⊥,ΨΨ}p<0.01 indicate significant differences of neutrophils, lymphocytes, and immature cells in relation to control (vehicle/saline) group, respectively. ^#,§p<0.05; ^##,§§p<0.01 indicate significant differences of neutrophils and lymphocytes in relation to vehicle/plus PQ group, respectively. ^*p<0.05 indicates significant difference of MPO activity in relation to control (vehicle/saline) group; ^#p<0.05 indicates significant difference to vehicle/plus PQ group (ANOVA followed by Bonferroni's post-hoc test).