Kynurenine Pathway as a New Target of Cognitive Impairment Induced by Lead Toxicity During the Lactation

Abstract

The immature brain is especially vulnerable to lead (Pb²⁺) toxicity, which is considered an environmental neurotoxin. Pb²⁺ exposure during development compromises the cognitive and behavioral attributes which persist even later in adulthood, but the mechanisms involved in this effect are still unknown. On the other hand, the kynurenine pathway metabolites are modulators of different receptors and neurotransmitters related to cognition; specifically, high kynurenic acid levels has been involved with cognitive impairment, including deficits in spatial working memory and attention process. The aim of this study was to evaluate the relationship between the neurocognitive impairment induced by Pb²⁺ toxicity and the kynurenine pathway. The dams were divided in control group and Pb²⁺ group, which were given tap water or 500 ppm of lead acetate in drinking water ad libitum, respectively, from 0 to 23 postnatal day (PND). The poison was withdrawn, and tap water was given until 60 PND of the progeny. The locomotor activity in open field, redox environment, cellular function, kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK) levels as well as kynurenine aminotransferase (KAT) and kynurenine monooxygenase (KMO) activities were evaluated at both 23 and 60 PND. Additionally, learning and memory through buried food location test and expression of KAT and KMO, and cellular damage were evaluated at 60 PND. Pb²⁺ group showed redox environment alterations, cellular dysfunction and KYNA and 3-HK levels increased. No changes were observed in KAT activity. KMO activity increased at 23 PND and decreased at 60 PND. No changes in KAT and KMO expression in control and Pb²⁺ group were observed, however the number of positive cells expressing KMO and KAT increased in relation to control, which correlated with the loss of neuronal population. Cognitive impairment was observed in Pb²⁺ group which was correlated with KYNA levels. These results suggest that the increase in KYNA levels could be a mechanism by which Pb²⁺ induces cognitive impairment in adult mice, hence the modulation of kynurenine pathway represents a potential target to improve behavioural alterations produced by this environmental toxin.

Figure 1

Lead concentration in different brain regions of mice at 23 PND, after Pb²⁺ exposure during lactation period. Determinations were made in hippocampus, striatum, cortex and cerebellum of mice. Data are mean ± S.E.M. T-test and Mann-Whitney test were used for comparisons, *P < 0.01 vs. each respective brain region control, n = 5 per group.

Figure 2

Locomotor activity of mice at 23 PND (A,B) and at 60 PND (C,D) in open field after Pb²⁺ exposure in drinking water during lactation period. Motor activity was assessed as the total distance walked (A,C) and the ambulatory time (B,D). Data are mean ± S.E.M. *P < 0.01 vs. control, T-test and Mann-Whitney test. N = 10–15 per group and per age.

Figure 3

Long-term memory evaluation after Pb²⁺ exposure during lactation period. Memory impairment in adult mice was evaluated through the distance traveled (B) and the time to reach target (C) 24 h after acquisition phase (A). Data are mean ± S.E.M. *P < 0.01 vs. control, T-test and Mann-Whitney test. N = 9–15 per group.

Figure 4

Kynurenine pathway alterations induced by Pb²⁺. (A) KAT II activity, (B) KYNA levels, (C) KMO activity and (D) 3-HK levels were evaluated in hippocampus, striatum, cortex and cerebellum of mice at 23 PND, after Pb²⁺ exposure during lactation period. Data are mean ± S.E.M. *P < 0.01 vs. each respective brain region control, T-test and Mann-Whitney test. N = 9 per group.

Figure 5

Long-term alterations on kynurenine pathway generated by Pb²⁺ treatment during lactation period of mice. (A) KAT II activity, (B) KYNA levels, (C) KMO activity and (D) 3-HK levels were evaluated in hippocampus, striatum, cortex and cerebellum of mice at 60 PND. Data are mean ± S.E.M. *P < 0.01 vs. each respective brain region control, T-test and Mann-Whitney test. N = 8 per group.

Figure 6

The lower triangular matrix contains the scatterplot for each pair of variables for both groups (control and Pb²⁺). The upper triangular matrix contains the Spearman’s rank correlation coefficient (r) and its associated p-value (p) calculated for each group.

Figure 7

Effect of Pb²⁺ treatment during lactation on the proportion of KAT II + and KMO + brain cells. Expression of KAT II (A) and KMO (C) was determined in the brain of mice at 60 PND. Number of positive cells to KATII (B) and KMO (D) were evaluated in the same samples. Data are mean ± S.E.M. *P < 0.05 vs. each respective brain region control, T-test and Mann-Whitney test.

Figure 8

Representative photomicrographs of hematoxylin-eosin-stained hippocampus sections (100x) of control and Pb²⁺ groups. (A) Control group, normal cells exhibited round-shaped and pale-stained nuclei. (B) Pb²⁺ group, dead cells exhibited a shrunken cytoplasm with pyknotic nuclei (black arrows). (C) Percentage of cellular damage in the hippocampal region of mice exposed to Pb²⁺. Values are expressed as the percentage of damaged cells counted in ten fields per area, in three slides per mouse. *P < 0.001 vs. control, T-test and Mann-Whitney test.