Differences in lead isotopic fingerprints between blood, hair and organs in lead-poisoned rats

Lead isotopic ratios of biosamples such as blood, hair and teeth are used as “fingerprints” to trace and identify sources of lead pollution in the environment. However, the reliability of this technique has not been confirmed. In previous studies, we investigated the differences in lead isotopic ratios between blood, hair, and organs from the same human body. To explore the relationship between isotopic ratios in environmental lead and biosamples, and to investigate the influence of exposure dose, rats were exposed to 0, 30, 60, and 120 mg/kg body weight of lead acetate every day for 4 weeks by intragastric administration. Lead isotopic ratios of blood, hair, liver, kidney and femur in rats, as well as that of the test substance lead acetate, were measured using inductively coupled plasma mass spectrometry. There were significant differences (P<0.05) in lead isotopic fingerprints between blood, hair, and tissues. Lead fingerprints of blood were influenced by lead exposure dose, while those of other tissues and organs were generally not. Its reliability and availability indicated that blood, but not hair, was thus a suitable biomarker for environmental lead exposure. The results of this study provide the scientific rationale for the use of lead isotopic ratios for the identification of human lead pollution sources.

Despite decades of study and regulation, lead poisoning remains a significant public health problem, especially in developing countries such as China. Identification of potential lead sources and prevention of lead pollution thus remain critically urgent tasks. Lead has four isotopes; 204 Pb, 206 Pb, 207 Pb, and 208 Pb. The composition of lead is largely unaffected by physical or chemical fractionation processes, and lead sources can therefore be identified by their isotopic "fingerprints" made up of characteristic ratios of the four isotopes. Compared with traditional epidemiologic methods, identification of lead sources based on isotopic ratios is faster, more accurate and less costly, and is therefore becoming a powerful complementary tool for determining potential lead sources. Many studies have investigated lead isotopic ratios in biological samples such as blood and/or hair to identify potential environmental lead sources [1][2][3][4][5][6][7][8], but the reliabilities of these different biological biomarkers have not yet been reported.
We previously demonstrated differences in isotopic fingerprints between blood, hair, and organs from the same individual [9,10]. In this study we aimed to explore the relationship between isotopic ratios in environmental lead and in different biosamples. Rats were subjected to lead poisoning and the isotopic ratios in blood, hair, and organs, together with those of the lead solution, were measured using inductively coupled plasma mass spectrometry (ICP-MS). This preliminary study aimed to confirm that differences in lead isotopic ratios exist among blood, hair, and other organs; to determine if isotopic ratios in biosamples are affected by lead exposure dose; and to identify the most reliable and suitable source of biosamples for tracing environmental lead sources.

Reagents
The soluble lead salt, lead acetate (Pb(CH 3 COO) 2 ·3H 2 O) (AR grade) was used in all experiments. The certified lead isotopic reference material NIST SRM 981 was used for correction of isotopic ratio measurements. Nitric acid (E-Pure grade, 71%), hydrogen peroxide (E-Pure grade, 30%) and perchloric acid (GR grade, 70%-72%), were used for digestion of biological samples. Ultra-pure water 18 MΩ cm −1 was used as a blank and to prepare the solutions.

Animal experiments
Physically healthy, male Sprague-Dawley rats, approximately 4 weeks old and weighing 80-100 g, were obtained from the Department of Laboratory Animal Science, Peking University Health Science Center. All the animals were maintained on a standard SPF laboratory low-lead diet, under the same standard barrier conditions. After adaptive feeding for 1 week, the rats were randomly separated into one control and three experimental groups, with eight rats in each group. The experimental groups consisted of low-, median-, and high-dose groups that received 30, 60, 120 mg/kg body weight of lead acetate every day for 4 weeks, respectively. Aqueous lead acetate solution was administered orally once a day at 1 mL/100 g body weight, using a plastic syringe with a catheter. The control group received ultra-pure water. The dorsal hair was shaved from the rats prior to the start of the experiment.
After 4 weeks of lead exposure, the rats were anesthetized by ether inhalation and killed by exsanguinations. The dorsal hair was shaved again and blood was collected into low-lead lithium heparin tubes. Liver, kidney, and femur were collected and weighed after elimination of connective and adipose tissue. All samples were stored at −20°C.
All animal procedures were carried out by licensed investigators and in accordance with the regulations of the Animals Act.

Sample preparation
Hair, blood and organ samples were wet digested in an open quartz system using a mixture of HNO 3 and HClO 4 (10:1 by volume). After 12 h cold digestion, the quartz vials then were progressively heated on a hot plate to 100, 120, 140, 160 and 180°C, until white vapor was given off. Ultrapure water was then added to drive off residual acid. The residue was diluted before analysis.

Measurements
Lead isotopic ratios were estimated using ICP-MS with Elan DRCII (PerkinElmer Instruments(Shanghai) Co. Ltd). Four lead isotopes 204 Pb, 206 Pb, 207 Pb, and 208 Pb were measured using the optimized parameters for the method: radio frequency power 1100 W, plasma gas flow rate 15 L/min, auxiliary gas flow rate 1.85 L/min, nebulizer gas flow rate 0.95 L/min, lens voltage 6.1 V , plus stage voltage 1100 V, peak hope transient scanning mode at one point scans, sweep 150/reading, dwell time 30 ms, and nine replicates. The relative standard deviations (n=7) of 10 ng/mL lead standard solution for 208 Pb/ 206 Pb, 207 Pb/ 206 Pb, and 204 Pb/ 206 Pb were 0.12%, 0.13%, and 0.27% under these optimization conditions.
The lead signal was corrected for mercury using the 204 Hg signal. Before the measurements the instrument had undergone a routine conditioning and testing procedure for 1-2 h. Lead isotopic certified reference material NIST SRM 981was used for isotope signal normalization. The common analyte internal standardization method 11,12 was applied for non-spectroscopic matrix effects and instrumental drift.

Statistical analysis
Statistical analyses were performed using SPSS version 14.0. One-way ANOVA was used to compare isotopic ratios between different dose groups, and paired t tests were used to compare isotopic ratios between blood, hair, and different organs.

Comparison of lead fingerprints among different dose groups
The lead isotope ratios in the different dose groups are compared in Tables 1-3. One-way ANOVA was used with a significance level of P = 0.05.
Significant differences in 208 Pb/ 206 Pb ratios were found between the low-and median-dose groups for blood (P < 0.01), the low-and high-dose groups for blood (P < 0.05); the low-and median-dose groups for liver (P < 0.05), and the median-and high-dose groups for liver (P < 0.01). There were no significant differences (P > 0.05) between any dose groups for kidney, femur or hair (Table 1).
There were no significant differences in 207 Pb/ 206 Pb ratios between any of the dose groups for all the measured biosamples ( Table 2).
There were significant differences in 204 Pb/ 206 Pb ratios only in kidneys between the low-and medium dose groups (Table 3).
Of the total of 45 comparisons from three dose groups, five different organs, and three isotopic ratios, only five demonstrated significant differences (P > 0.05), indicating that lead fingerprints in the body remain relatively stable. The 207 Pb/ 206 Pb ratio was the most stable, and showed no differences between dose groups or biosamples (P < 0.05). The femur provided the most stable biosample in terms of isotopic ratios, with no significant differences between dose   groups for any of the lead isotope ratios. Statistical analysis demonstrated that blood lead isotopic ratios varied with exposure dose, while the lead fingerprints of other organs generally did not. The blood lead isotopic ratio in the low-dose group differed from that of the test substance, but the difference decreased as the dose increased, and disappeared in the high-dose group.
The results of this study suggest that: (a) Blood may act as the first barrier to lead exposure in the body, and is thus likely to play a role in the selective accumulation or fractionation of lead isotopes, whilst possessing a threshold for selective accumulation or fractionation. As concentration of the test substance increases, the threshold is exceeded, and the difference in lead isotope ratios between the blood and the test substance declines. (b) Blood transports the lead to various tissues and organs. When the lead, which has already been buffered by the blood, arrives at the tissues and organs it accumulates there and becomes relatively stable, with its isotopic ratios relatively unaffected by the test substance dose. Further studies are needed to verify these assumptions.

Comparison of lead isotopic fingerprints between blood, hair and organs
Comparisons of lead isotope ratios between blood, hair, and organs are presented in Tables 4-6. Where lead fingerprints were not significantly different between dose groups, data a) ** indicates P<0.01, * indicates P<0.05, "-" indicates lead isotope ratios in this organ were not influenced by dose, and dose groups were therefore combined.   for all dose groups were combined.
There were significant differences in 208 Pb/ 206 Pb ratios between blood and liver, blood and kidney, and blood and bone, and between any two of kidney, bone and hair. There were no significant differences between hair and blood, or hair and liver (Table 4).
There were significant differences (P < 0.05) in 207 Pb/ 206 Pb ratios between any two biosamples, apart from blood and bone, blood and hair, and liver and kidney (Table 5).
There were significant differences in 204 Pb/ 206 Pb ratios between any two biosamples, apart from blood and liver, kidney and femur, kidney and hair, and femur and hair ( Table 6).
Thus among the 10 biosample comparisons, there were significant differences in all three isotopic ratios for two (blood-kidney, and liver-femur), in two isotopic ratios for six pairs, and in one isotopic ratio for two pairs (blood-hair, and liver-kidney). These results indicate that general differences in lead isotopic ratios exist among biosamples in the body, and that the samples can be distinguished by at least one lead isotopic ratio. The presence of differences among biosamples in rats is in accord with the results from humans observed in our earlier research.
The current study provides the first evidence of differences among biosamples in terms of lead isotope ratios in animals. However, the mechanism responsible for this difference remains unclear, and suitable reference materials are currently extremely rare. Further studies are therefore needed to confirm the above assumptions.

Comparison of lead isotopic fingerprints between biosamples and test substance
Comparisons of lead isotopic ratios between biosamples and the test substance for all experimental groups and for separate dose groups are presented in Figures 1 and 2, respectively.
It is interesting to note that the lead isotopic ratios of  blood, hair and organs are distributed within different elliptical areas around the test substance ( Figure 1). Relative to the test substance, liver ratios are distributed in the upper left region, kidney ratios in the lower left region, and femur ratios in the lower right region. In contrast, hair ratios are distributed in the lower right region, furthest from the test substance, while blood ratios are distributed within an elliptical area with the test substance at the center. The scattered points for blood and liver become more centralized with increasing dose (Figure 2). In the high-dose group, blood lead isotopic ratios are distributed more densely and closely around the test substance, compared with other tissues.
It is likely that some bio-fractionation occurs in tissues and organs after the lead is taken up by the rats, and the lead isotopic ratio of some samples differs from that of the isotopic ratio of the test substance. Hair is thus unsuitable as a biomarker, while the reliability and availability of blood make it better suited as a biomarker for tracing environmental lead exposure, especially under high-dose conditions.

Conclusions
(1) There are differences in lead isotopic ratios between blood, hair and other organs in lead-poisoned rats.
(2) Blood lead isotopic ratios are influenced by lead exposure dose, while those of other tissues are generally not.
(3) Blood is more suitable than hair as a biomarker for identifying environmental lead sources.
(4) This represents the first report of differences in lead isotopic ratios between blood, hair and organs in rats, but further research is needed to confirm these results.