1. Boris A. Katsnelson, Ekaterina P. Kireyeva, Sergey V. Kuzmin, Larissa I. Privalova, Nina A. Khrushcheva, Yaakov B. Beikin, Tatiana V. Postnikova, Natalya S. Zhuravleva, Nina P. Makarenko, Tamara D. Degtyareva, Anastasia V. Porovitsina Ekaterinburg Medical Research Center for Prophylaxis and Health Protection in Industrial Workers; The Urals Medical Academy; Center for Laboratory Diagnostics of Mother’s and Children’s Diseases - Ekaterinburg, Russia AN ASSOCIATION BETWEEN RENAL DAMAGE AND URINARY CADMIUM AND LEAD LEVELS IN YOUNG RUSSIAN CHILDREN: EPIDEMIOLOGY AND INTERVENTION

2. OBJECTIVES: (a) to find out whether an incipient renal damage in children dwellers of urban areas in an industrial region could be associated with the combined toxicity of cadmium and lead; (b) to test if such a damage can be beneficially influenced by a combination of bio-protective agents previously proven in an animal experiment to be inhibitors of cadmium/lead nephrotoxicity.

3. Stage I ANALYTICAL EPI-STUDY

4. In 2004 and 2005 we examined 3 to 7 years old preschool children from four towns in the Middle Urals, three of them being polluted by the copper industry. The location of those towns within the boundaries of the Sverdlovsk Oblast’ (together with its administrative center, city of Ekaterinburg) as well as the location of this Oblast’ within the Russian Federation are shown on the maps.

5. Sverdlovsk Region Moscow

7. Only those children were included into the studied groups who had no known renal disease. Urinary concentrations of Cd and Pb, and of beta-2- microglobulin (B2u), along with standard clinical laboratory indices, were measured in 89 children in the 2004 (Sredneuralsk – Sysert) cohort, and 184 in the 2005 (Pervouralsk – Revda) cohort. A case-control study design was used to evaluate the association between B2u and body burdens of Pb and Cd as measured by their urinary levels. “Cases” were defined as those with B2u levels above the median and “controls” as those at or below the median. B2u levels ranged from 10 to 1200 ug/L. In both studies mean concentrations of both metals proved higher in cases as compared with controls.

8. Mean urinary concentrations of beta-2 microglobulin, Cadmium and Lead Subgroup of the childre n Mean urine concentrations (±s.e.), μ g/L B2uCadmiumLead The 1 st study *) “Cases”227±281.31±0.2623.73±3.4 3 “Controls”61±40.75±0.1312.71±2.0 1 t =5.781.952.77 The 2 nd study** ) “Cases”241.5±19.76.5±0.762.1±2.7 “Controls”82.1±3.04.9±0.641.9±2.5 t =8.01.95.5 *) Sredneuralsk and Sysert; median B2u level = 100 μg/L. **) Pervouralsk and Revda; median B2u level =120 μg/L.

9. Associations between Cd or Pb levels and the probability of a child being classified as a case (odds ratios adjusted for area, gender, town, and individual risk factors proved statistically significant in univariate regression analysis) Metal2004 study2005 study OR per 1 ug/L (95% CI) P<OR per 1 ug/L (95% CI) P< Cadmium1.89 (1.13- 3.16) 0.021.13 (1.04- 1.22) 0.03 Lead1.19 (1.07- 1.31) 0.00 1 1.03 (1.02- 1.05) 0.001

10. Stage II ANIMAL EXPERIMENTAL STUDY

11. In experiments on rats it was shown that: Both Cadmium and Lead, when administered repeatedly in low doses as single toxics or in a dual combination, cause renal damage manifested with a lot of functional, biochemical and morphometrical indices; In doses equivalent as fractions of respective acute LD50, sub-chronic nephrotoxicity of Lead is higher as compared with that of Cadmium; A complex of bio-protectors comprising pectin, glutamate, a multivitamin-multi-mineral preparation, and a calcium supplement attenuates the combined nephrotoxicity of these metals.

12. Stage III AN INTERVENTIONAL EPI-STUDY

13. In 2006 we examined 38 children aged 3-7 attending kindergartens in the town of Krasnouralsk polluted with emissions from a big copper smelter. This time children with renal disturbances have not been excluded. Concentrations of cadmium, lead, and beta-2 microglobulin (B2u) were measured in their morning urine samples, along with liquid crystallography and standard clinical laboratory indices. All the tests were performed just before and immediately after the 5 weeks period during which the children were being given (with the informed consent of their parents, and under medical supervision) the bio-protective complex previously tested in our animal experiment.

14. The initial B2u levels in this group ranged from 20 to 3180 ug/L. Only 5 children (13.2%) had levels >300 ug/L. However, many children were found to have other renal abnormalities: Micro-urolithiasis seen with the polarization microscopy (in 86.8% of children) which revealed also the formation of birefringent lipoprotein crystals characteristic of renal membranolysis of different extent (in 100%) and the presence of an albuminous ring around the microscopy preparation (in 65.7%). One third of the group had abnormally high urine relative density (> 1025). After the bio-protective intervention the prevalence of all these indices has statistically significantly (p<0.05) diminished. Metals and B2u levels has diminished as well.

15. The mean concentrations (ug/L ±s.e.) of B2u, Cd and Pb in the group of Krasnouralsk children IndexInitial measure Final measure Beta-2 microglob uline 216.6±85.794.5±16.7 Cadmium0.22±0.20.11±0.01 Lead15.23±1.1810.20±1.35

16. CONCLUSIONS Urinary cadmium and lead concentrations characteristic of children living in polluted areas may be – at least partially – associated with an incipient renal damage in some of them. A beneficial renal effect produced in children by a combination of bio-protective agents previously proven in an animal experiment to be inhibitors of cadmium/lead nephrotoxicity may be considered as additional circumstantial evidence of the said association.