José Paulo Santos

Lead is a potent toxicant associated with adverse cardiovascular effects and hypertension in children. Yet, few studies have determined if autonomic dysfunction associated with lead exposure involves brain regions which regulate autonomic responses. Central autonomic nuclei such as the nucleus tractus solitarius (NTS) and hypothalamic defence area (HDA) may be particularly sensitive to lead infiltration because they are adjacent to ventricles and areas with semi-permeable blood-brain-barriers. To understand if autonomic nuclei are sensitive to lead accumulation Wistar rats were exposed to lead from the gestational period and lead levels were quantified in brain regions that regulate arterial pressure: the NTS and the HDA. Energy dispersive X-ray fluorescence (EDXRF) was used to quantify total brain lead levels and revealed no differences between exposed and control tissues; measured values were close to the detection limit (2μg/g). Electrothermal atomic absorption spectrometry (ETAAS) was also used, which has a greater sensitivity, to quantify lead. There was ∼2.1μg/g lead in the NTS and ∼3.1μg/g lead in the HDA of exposed rats, and no lead in the control rats. There were greater lead levels in the HDA (∼50%) as compared with the NTS. Pathology studies revealed more prominent lead granules in the HDA as compared with the NTS. Increased microglia and astrocyte activation was also noted in the NTS of lead exposed rats as compared with the HDA. Regional differences in neuro-inflammatory responses likely contribute to heterogeneous lead accumulation, with enhanced clearance of lead in the NTS. Future studies will resolve the mechanisms underpinning tissue-specific lead accumulation.

Lead is a potent toxicant associated with adverse cardiovascular effects and hypertension in children. Yet, few studies have determined if autonomic dysfunction associated with lead exposure involves brain regions which regulate autonomic responses. Central autonomic nuclei such as the nucleus tractus solitarius (NTS) and hypothalamic defence area (HDA) may be particularly sensitive to lead infiltration because they are adjacent to ventricles and areas with semi-permeable blood-brain-barriers. To understand if autonomic nuclei are sensitive to lead accumulation Wistar rats were exposed to lead from the gestational period and lead levels were quantified in brain regions that regulate arterial pressure: the NTS and the HDA. Energy dispersive X-ray fluorescence (EDXRF) was used to quantify total brain lead levels and revealed no differences between exposed and control tissues; measured values were close to the detection limit (2μg/g). Electrothermal atomic absorption spectrometry (ETAAS) was also used, which has a greater sensitivity, to quantify lead. There was ∼2.1μg/g lead in the NTS and ∼3.1μg/g lead in the HDA of exposed rats, and no lead in the control rats. There were greater lead levels in the HDA (∼50%) as compared with the NTS. Pathology studies revealed more prominent lead granules in the HDA as compared with the NTS. Increased microglia and astrocyte activation was also noted in the NTS of lead exposed rats as compared with the HDA. Regional differences in neuro-inflammatory responses likely contribute to heterogeneous lead accumulation, with enhanced clearance of lead in the NTS. Future studies will resolve the mechanisms underpinning tissue-specific lead accumulation.

An ultrasonic assisted solid‚Äìliquid extraction method was developed to determine the level of lead in the brain and urine of rats. Lead was determined by electrothermal atomic absorption spectrometry with longitudinal-Zeeman background correction. Several analytical drawbacks were addressed and overcome, namely small brain sample mass and the formation of precipitate in the urine samples. Utrasonication provided by an ultrasonic probe succeeded in extracting lead from brain samples. Furthermore, it was demonstrated that the formation of a precipitate lowered the lead content in the liquid phase of the urine. Lead was back extracted from the precipitate to the liquid phase with the aid of ultrasonic energy and acidifying the urine with 10% v/v nitric acid. A microwave-assisted acid digestion protocol was used to check the completeness of the lead extraction. The within bath and between bath precision was 5% (n = 9) and 7% (n = 3) respectively. The limit of quantification was 1.05 Œºg g‚àí1 for brain samples and 2.1 Œºg L‚àí1 for urine samples. A total of 6 samples of urine and 12 samples of brain from control rats and another 6 samples of urine and 12 samples of brain from rats fed with tap water rich in lead acetate were used in this research. Lead levels in brain and urine from exposed rats ranged from1.9 ¬± 0.2 Œºg g‚àí1 to 3.5 ¬± 0.2 Œºg g‚àí1 and from 752 ¬± 56 Œºg L‚àí1 to 60.9 ¬± 1.2 mg L‚àí1 respectively. Statistically significant differences of levels of lead in brain and urine were found between exposed and non exposed rats.

Talanta, 155 + (2016) 107-115. doi:10.1016/j.talanta.2016.04.028

The accumulation of lead in several bones of Wistar rats with time was determined and compared Q3 for the different types of bones. Two groups were studied: a control group (n = 20), not exposed to lead and a contaminated group (n = 30), exposed to lead from birth, first indirectly throughmother’s milk, and then directly through a diet containing lead acetate in drinking water (0.2%). Rats age ranged from 1 to 11 months, with approximately 1 month intervals and each of the collections had 3 contaminated rats and 2 control rats. Iliac, femur, tibia–fibula and skull have been analysed by energy dispersive X-ray fluorescence technique (EDXRF). Samples of formaldehyde used to preserve the bone tissues were also analysed by Electrothermal Atomic Absorption (ETAAS), showing that there was no significant loss of lead from the tissue to the preservative. The bones mean lead concentration of exposed rats range from 100 to 300 mg g 1 while control rats never exceeded 10 mg g 1. Mean bone lead concentrations were compared andthe concentrations were higher in iliac, femur and tibia–fibula and after that skull. However, of all the concentrations in the different collections, only those in the skull were statistically Q4 significantly different (p o 0.05) from the other types of bones. Analysis of a radar chart also allowed us to say that these differences tend to diminish with age. The Spearman correlation test applied to mean lead concentrations showed strong and very strong positive correlations betweenall different types of bones. This test also showed that mean lead concentrations in bones are negatively correlated with the age of the animals. This correlation is strong in iliac and femur and very strong in tibia–fibula and skull. It was also shown that the decrease of lead accumulation with age is made by three plateaus of accumulation,

The accumulation of lead in several bones of Wistar rats with time was determined and compared Q3 for the different types of bones. Two groups were studied: a control group (n = 20), not exposed to lead and a contaminated group (n = 30), exposed to lead from birth, first indirectly through
mother’s milk, and then directly through a diet containing lead acetate in drinking water (0.2%). Rats age ranged from 1 to 11 months, with approximately 1 month intervals and each of the collections had 3 contaminated rats and 2 control rats. Iliac, femur, tibia–fibula and skull have been analysed by energy dispersive X-ray fluorescence technique (EDXRF). Samples of formaldehyde used to preserve the bone tissues were also analysed by Electrothermal Atomic Absorption (ETAAS), showing that there was no significant loss of lead from the tissue to the preservative. The bones mean lead concentration of exposed rats range from 100 to 300 mg g 1 while control rats never exceeded 10 mg g 1. Mean bone lead concentrations were compared and
the concentrations were higher in iliac, femur and tibia–fibula and after that skull. However, of all the concentrations in the different collections, only those in the skull were statistically Q4 significantly different (p o 0.05) from the other types of bones. Analysis of a radar chart also allowed us to say that these differences tend to diminish with age. The Spearman correlation test applied to mean lead concentrations showed strong and very strong positive correlations between
all different types of bones. This test also showed that mean lead concentrations in bones are negatively correlated with the age of the animals. This correlation is strong in iliac and femur and very strong in tibia–fibula and skull. It was also shown that the decrease of lead accumulation with age is made by three plateaus of accumulation,

The concentration of lead in liver and kidneys of Wistar rats, fed with lead since fetal period in relation to their age and to a control group, was determined. A group of rats was exposed to lead acetate (n=30) in drinking water and the other group was exposed to normal water (n=20). Samples were collected from rats aging between 1 and 11 months and were analyzed by Energy Dispersive X-ray Fluorescence (EDXRF) without any chemical preparation. The EDXRF results were assessed by the PIXE (Proton Induced X-ray Emission) technique. The formaldehyde used to preserve the samples was also analyzed by ETAAS (Electro-Thermal Atomic Absorption Spectrometry) in order to verify if there was any loss of lead from the samples to the formaldehyde. We found that the loss was not significant (<2%). Concerning the mean values of the lead concentration measured in the contaminated soft tissues, in liver they range from 6 to 22μgg(-1), and in kidneys from 44 to 79μgg(-1). The control rats show, in general, values below the EDXRF detection limit (2μgg(-1)). The ratio kidney/liver ranges from 2 to 10 and is strongly positively correlated with the age of the animals. A Spearman correlation matrix to investigate the correlation between elemental concentrations and the dependence of these concentrations with age showed that there is a strong positive correlation with age for lead in the liver but not in the kidney. The correlation matrix showed also that the concentration of lead in these two soft tissues is not correlated. The lead accumulation in liver is made by different plateaus that strongly decrease with age. It was verified the existence of two levels of accumulation in kidney, not very highlighted, which might be indicative of a maximum accumulation level for lead in kidney.

An ultrasonic assisted solid–liquid extraction method was developed to determine the level of lead in the brain and urine of rats. Lead was determined by electrothermal atomic absorption spectrometry with longitudinal-Zeeman background correction. Several analytical drawbacks were addressed and overcome, namely small brain sample mass and the formation of precipitate in the urine samples. Utrasonication provided by an ultrasonic probe succeeded in extracting lead from brain samples. Furthermore, it was demonstrated that the formation of a precipitate lowered the lead content in the liquid phase of the urine. Lead was back extracted from the precipitate to the liquid phase with the aid of ultrasonic energy and acidifying the urine with 10% v/v nitric acid. A microwave-assisted acid digestion protocol was used to check the completeness of the lead extraction. The within bath and between bath precision was 5% (n&#xa0;=&#xa0;9) and 7% (n&#xa0;=&#xa0;3) respectively. The limit of quantification was 1.05&#xa0;μg&#xa0;g−1 for brain samples and 2.1&#xa0;μg&#xa0;L−1 for urine samples. A total of 6 samples of urine and 12 samples of brain from control rats and another 6 samples of urine and 12 samples of brain from rats fed with tap water rich in lead acetate were used in this research. Lead levels in brain and urine from exposed rats ranged from1.9&#xa0;±&#xa0;0.2&#xa0;μg&#xa0;g−1 to 3.5&#xa0;±&#xa0;0.2&#xa0;μg&#xa0;g−1 and from 752&#xa0;±&#xa0;56&#xa0;μg&#xa0;L−1 to 60.9&#xa0;±&#xa0;1.2&#xa0;mg&#xa0;L−1 respectively. Statistically significant differences of levels of lead in brain and urine were found between exposed and non exposed rats.