Effects of sodium para-aminosalicylic acid on chelation treatment in Pb-exposed mice

Lead (Pb) is a corrosion-resistant, heavy, non-ferrous metal. Several metal chelators have been used for the treatment of Pb poisoning. However, the efficacy of sodium para-aminosalicylic acid (PAS-Na) in enhancing Pb excretion has yet to be fully characterized. Healthy male mice (90) were divided into six groups, the normal control group was intraperitoneally (i.p.) injected with saline and the remaining group of mice i.p. 120 mg/kg Pb acetate. Four hour later, mice were subcutaneously (back) injected (s.c.) with (80, 160, 240 mg/kg) PAS-Na or 240 mg/kg edetate calcium disodium (CaNa2EDTA) or an equivalent amount of saline, once per day for 6 days. After 24-h urine sample collections, the animals were anesthetized with 5% chloral hydrate and sacrificed in batches on the 2nd, 4th, or 6th day. Levels of Pb [including manganese (Mn) and copper (Cu)] in the urine, whole blood, and brain tissues were analyzed by graphite furnace atomic absorption spectrometry. The results showed that Pb exposure increased its levels in urine and blood, and PAS-Na treatment may afford antagonistic effect on Pb poisoning, suggesting that PAS-Na is a potentially effective treatment to promote excretion of Pb.


Introduction
Lead (Pb) is an environmentally polluting heavy metal. Exposure to Pb in industry and the workplace has become a growing health concern in recent years (Lu et al., 2022;Kan et al., 2021;Luo et al., 2016;He et al., 2017). Pb enters the body predominantly through the respiratory and digestive tracts, and it can cause harmful effects to the nervous, digestive, and hematopoietic systems (Charkiewicz and Backstrand, 2020;Reuben, 2018).
In 1950, the US Food and Drug Administration (FDA) approved CaNa 2 EDTA to treat Pb poisoning patients (Flora and Pachauri, 2010). CaNa 2 EDTA is not metabolized, and it can redistribute Pb to the brain following acute poisoning (Gerhardsson and Kazantzis, 2015). CaNa 2 EDTA has been used for the treatment of Pb poisoning, CaNa 2 EDTA does not cross the blood-brain barrier. However, it can cause a reduction in peripheral blood Pb levels. CaNa 2 EDTA can indirectly promote the excretion of brain Pb via urine.
Experimental and clinical application of paraaminosalicylic acid (PAS) or PAS-Na is effective in treating Mn poisoning, likely related to the low molecular weight characteristics of PAS or PAS-Na (Jiang et al., 1992(Jiang et al., , 2006Tandon et al., 1975;Tandon, 1978;1 Zheng et al., 2009). PAS-Na and its main metabolite, N-acetyl-para-aminosalicylic acid (AcPAS), readily cross the blood-brain barrier, and PAS and AcPAS are effective in reducing brain Mn levels (Hong et al., 2011). It has been shown that short-term Mn exposure significantly increased Mn concentrations in plasma, red blood cell (RBC), cerebrospinal fluid (CSF), brain, and soft tissue of rats. Treatment with PAS (200 mg/ kg) for 3 weeks significantly reduced Mn levels in liver, heart, spleen, and pancreas of Mn-treated rats by 25-33%, and after 6 weeks of treatment, the Mn levels in the striatum, thalamus, choroid plexus, hippocampus, and frontal cortex were decreased by 16-29% (p < 0.05). It was suggested that PAS may act as a chelating agent to remove Mn from tissues (Zheng et al., 2009).
Brain Mn and brain Cu levels were increased in Mntreated rats, and PAS-Na treatment reduced brain Mn and brainstem Cu levels. (Jiang et al., 1992). Increased Pb burden in Pb-exposed workers has also been shown to increase Cu levels (Anetor et al., 2002). Pb exposure for 4 weeks significantly increased Pb content in rat hippocampus, and treatment with PAS-Na (80, 160, and 240 mg/kg) for 2 weeks significantly reduced hippocampal Pb levels .
Pb can also activate the ERK1/2-p90RSK/NF-κB pathway and increase the level of the inflammatory cytokines, IL-1β, in rat hippocampus. PAS-Na can restore or ameliorate Pb-induced learning and memory deficits, secondary to reduction in hippocampal Pb levels and induction of anti-inflammatory effects (Lu et al., 2022;Li et al., 2022;Zhao et al., 2022). Therefore, herein, we investigated the effects of PAS-Na on the excretion of Pb, Cu, and Mn from brain, blood, and urine of mice exposed to Pb for a shortterm, to provide a scientific basis for the clinical application of PAS-Na in the treatment of Pb poisoning.

Animal experiments
A total of 90 CL grade male mice were purchased from the Experimental Animal Center of Guangxi Medical University [SCXK (Gui) 2020-11010]. All experimental procedures were approved by the Animal Ethics Committee of Guangxi Medical University and performed following their guidelines. Mice were maintained in conditions of temperature (22 ± 3°C) and humidity (50 ± 10%) with a 12 h light/12 h dark cycle. Food and distilled water were available ad libitum.
After a 7-day acclimatization period, 90 adult (weighing 18-25 g) Kunming mice were divided into a normal control group, Pb-exposed group, PAS-Na (80, 160, 240 mg/kg, referred to L-, M-, H-PAS) treatment groups, and CaNa 2 EDTA (240 mg/kg) positive treatment group, 5 mice per group. The normal control group was intraperitoneally (i.p.) injected with saline and the remaining group of mice i.p. Pb acetate (120 mg/kg). Four hour later, mice were subcutaneously (back) injected (s.c.) with (L-, M-, H-) PAS-Na or CaNa 2 EDTA or an equivalent amount of saline, once a day, for 6 days. Body weights were recorded daily. Based on our earlier study, Pb exposure and PAS-Na therapy doses were selected (Deng et al., 2009).

Collection of animal blood, urine, and brain tissues
At the end of the experiment, mice were placed in the metabolic cage; food and distilled water were available ad libitum. After collection of 24-h urine, the mice were anesthetized with 5% chloral hydrate (0.1 mL/10 g body weight, i.p.) and sacrificed in batches on the 2nd, 4th, and 6th day. Next, whole blood and brain tissues were collected. Brains were weighed and stored at À80°C for metal content analyses. To prevent metal contamination, samples were stored in trace element-free tubes.

Pb, Mn, and Cu detection
Pb, Mn, and Cu assays were performed by graphite furnace atomic absorption spectrometry (AAS) with AAnalyst 800 spectrometers (PerkinElmer, USA) using a Zeeman background correction. The light source consisted of a hollow cathode lamp. Furnaces with integrated platform were used.
Analysis of Pb, Cu, and Mn in blood, urine, and brain Brain tissue samples were digested by microwave at 190°C with 5 mL ultrapure 65% HNO 3 . Subsequently, samples were placed on 150°C graphite heating plates to remove the acid for 90 min. Samples were evaporated to approximately 0.5 mL, and cooled down to room temperature. All brain samples were diluted to 5 mL with distilled water. The samples of blood and urine were analyzed absent digestion. Lastly, Pb, Mn, and Cu levels in blood, urine, and brain tissues samples were analyzed with AAnalyst 800 AAS (PerkinElmer, USA). Blank and quality controls were processed simultaneously.

Statistical analysis
All data analysis was performed with the Statistical Package for Social Sciences version 26.0 (SPSS Inc.), and all the figures were performed with the GraphPad Prism 9. Results are expressed as means ± SD of at least three independent experiments. Normality of distribution was assessed by the Lilliefors test, and homogeneity of variance was tested with the Levene's test. Statistical comparisons were performed by oneway analysis of variance (ANOVA).

Results
Effects of PAS-Na treatment on Pb levels in urine of Pb-exposed mice As shown in Figure 1, Pb levels were statistically significantly different between the Pb-exposed and the three PAS-Na administered groups (L-PAS p < 0.05, M-PAS p < 0.01, H-PAS p <0.001 on the 2nd day. L-PAS p < 0.001, M-PAS p < 0.001, H-PAS p < 0.01 on the 3rd day. L-PAS p < 0.001, M-PAS p < 0.01, H-PAS p < 0.01 on the 4th day). The efficacy for increasing Pb levels in the PAS-Na group were lower than in the CaNa 2 EDTA group.
Effects of PAS-Na treatment on Pb levels in whole blood of Pb-exposed mice As shown in Figure 2, after L-, M-, or H-PAS-Na treatment, blood Pb levels were statistically significantly different from the Pb-exposed mice on day 2 (p < 0.05). Blood Pb level of Pb-exposed group compared with control group, the difference was statistically significant (p < 0.01). CaNa 2 EDTA treatment significantly reduced blood Pb levels compared with Pb alone exposed mice (p < 0.01) on the 6th day.
Effects of PAS-Na treatment on Mn levels in urine of Pb-exposed mice Urinary Mn levels were higher in the CaNa 2 EDTA treatment group than in the Pb-exposed group (p < 0.001) from the 2nd to the 6th day Figure 3.
Effects of PAS-Na treatment on Cu levels in urine of Pb-exposed mice As showed in Figure 4, the CaNa 2 EDTA treatment significantly reduced urinary Cu levels compared with Pb-exposed mice (p < 0.05) on the 2nd day.
Effects of PAS-Na treatment on Pb, Mn, Cu levels in brain of Pb-exposed mice Compared with the normal control group (0.20 ± 0.02 μg/g), the brain Pb levels in the Pb-exposed group (0.21 ± 0.08 μg/g) did not increase significantly and the difference was not statistically significant (p > 0.05). There was no statistically significant difference in brain Pb levels between the Pb-exposed group and the PAS-Na intervention group (0.22 ± 0.09 μg/g) (p > 0.05). The same results were found for Mn and Cu.

Discussion
The health hazards caused by environmental Pb exposure are of global concern (Shi et al., 2019). Once Figure 1. The effect of PAS-Na on urine Pb levels in mice. Data are mean ± SD, n = 5 for each group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Pb-exposed mice.
absorbed, Pb mainly distributes to blood and tissues, and is excreted in the urine (Shao et al., 2017). Pb levels in urine (Sakai, 2000) and blood (Shao et al., 2017;Mohammadyan et al., 2019) have been commonly used as biomarkers of contemporary Pb exposure in vivo. Pb levels in blood are regarded as valuable and predictive biomarkers of long-term Pb exposure and Pb-induced neurotoxicity (Li et al., 2023). To detect the early toxic effects of Pb, blood and urine Pb levels have been recommended as optimal surrogates (Shao et al., 2017). Therefore, this approach has been implemented herein.
According to the Diagnostic Criteria for Occupational Chronic Pb Poisoning (GBZ37-2015), urinary Pb ≥120 μg/L represent a diagnostic indicator of mild Pb poisoning. In this experiment, the mean urinary Pb level in Pb-exposed mice was 716.9 μg/L, suggesting that the dose and experimental duration were appropriate to mimic a poisoning level consistent with previously reported results (Deng et al., 2009).
Chelation therapy is often used to treat environmental, occupational, or life metal poisoning (Andersen, 1999;Aaseth et al., 2015). PAS-Na has shown efficacy as a Mn chelator, both in blood and brain (Yuan et al., 2016;Li et al., 2020). The rats were stained with Pb acetate (10 mg/ kg) for 4 weeks, and treated with PAS-Na (80, 160, 240 mg/kg) for 2 weeks. It was found that the Pb in hippocampus of rats was significantly reduced by PAS-Na, suggesting that PAS-Na can promote the excretion of Pb in hippocampus of rats . The results of Figure 3. The effect of PAS-Na on Mn levels in the urine in mice. Data are mean ± SD, n = 5 for each group; ***p < 0.001 vs. Pb-exposed mice. Figure 2. The effect of PAS-Na on blood Pb levels in mice. Data are mean ± SD, n = 5 for each group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. Pb-exposed mice.
our study showed that PAS-Na significantly increased murine urinary Pb levels, demonstrating a statistically significant difference compared with the control group. Furthermore, PAS-Na promoted faster urinary Pb excretion within 4 days of administration. In addition, PAS-Na increased blood Pb levels within 2 days of administration. However, no effect of PAS-Na on brain Pb level was observed, which may be related to the short duration of PAS-Na treatment in our experimental paradigm.
Homeostasis of metals plays an important role in maintaining optimal function (Mezzaroba et al., 2019). Both Mn and Cu are the most abundant bodily minerals, playing essential roles in maintaining normal functioning of the CNS (Chin-Chan et al., 2022;Ivleva et al., 2022) and other organs. Cu is essential for key biological functions such as hematopoiesis and iron metabolism, and increased Cu exposure can cause neurotoxicity (Uriu-Adams and Keen, 2005;Montes et al., 2014). The results reported herein show that upon acute Pb poisoning, no significantly statistical changes were ascribed to PAS-Na treatment on urinary or brain Mn and Cu levels. Our results also established that the PAS-Na group had lower Pb and Mn urinary excretion compared to the CaNa 2 EDTA group, and that CaNa 2 EDTA treatment reduced urinary Cu levels, suggesting that CaNa 2 EDTA showed greater efficacy in promoting urinary Pb, Mn, and Cu. Moreover, CaNa 2 EDTA treatment significantly reduced blood Pb levels compared with Pb-exposed mice on the 6th day after treatment, corroborating previous findings (Keshri et al., 2021;Li et al., 2017).
In summary, this novel study showed that PAS-Na led to a significant increase in urinary Pb excretion in an acute murine Pb-exposed model. However, PAS-Na showed no efficacy in attenuating brain Pb levels. To further characterize its efficacy in brain Pb chelation, it is imperative to increase the duration of treatment time with PAS-Na.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of Figure 4. The effect of PAS-Na on Cu levels in the urine of mice. Data are mean ± SD, n = 5 for each group; *p < 0.05, **p < 0.01 vs. Pb-exposed mice.
this article: This work was supported by the support was provided by grants from the National Natural Science Foundation of China (NSFC 81,773,476).

Ethics approval
All animal procedures performed in this study were performed strictly according to the international standards of animal care guidelines and have been approved by the Animal Care and Use Committee of Guangxi Medical University.

Data availability
All data generated or analyzed during this study are included in this published article.