Fd ('hem. 7bxic. Vol. 31, No. 10, pp. 689-699, 1993 0278-6915/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd Research Section EFFECTS OF DIETARY IRON AND FOLATE SUPPLEMENTATION ON THE PHYSIOLOGICAL CHANGES PRODUCED IN WEANLING RATS BY SODIUM SACCHARIN EXPOSURE E. M. GARLAND*, R. SHAPIROt, J. M. WEHNER, L. S. JOHNSON, B. J. MATTSON, M. KHACHAB, M. ASAMOTO and S. M. COHEN Departments of Pathology and Microbiology and the Eppley Institute, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-3135, USA (Accepted 14 April 1993) Abstract--Exposure of rats to high dietary levels of sodium saccharin (NaSac) started in utero produce physiological effects at 30 days post-birth that are similar to those found in pups of iron-deficient dams. These similarities suggest that some of the changes due to NaSac are secondary to iron deficiency. The present experiment investigated whether the effects of 7.5% dietary NaSac in the newborn rat could be prevented by dietary iron and/or folate supplementation. The NaSac-related effects prevented by iron supplementation included anaemia, decreased serum iron and folate, increased serum cholesterol and triglyceride and increased serum vitamin E. Folate supplementation prevented NaSac-induced depression of serum folate and increase in serum vitamin E. Although bladder hyperplasia was increased by dietary iron and/or folate supplementation, the majority of the urinary chemistry changes associated with NaSac treatment were not affected. The results show that some physiological changes associated with NaSac treatment in the newborn rat may occur as a consequence of iron deficiency rather than a direct effect of NaSac treatment. These changes may be independent of the urinary and bladder effects, which are not reversed by iron supplementation. INTRODUCTION Sodium saccharin (NaSac) causes bladder tumours when fed at high doses (/> 3% of the diet) to male rats at birth, or in utero, and throughout their lifetime. If feeding is initiated at approximately 6 wk of age, proliferative changes occur in the bladder within 10 wk, but generally no significant incidence of blad- der tumours is produced after 2 yr. In previous studies conducted to determine the effects of high dietary NaSac (7.5%) administered during the neonatal period on physiological and nutritional parameters that might be associated with the subsequent incidence of bladder tumours, we observed depressed growth, anaemia, decreased iron concentration in serum, decreased serum and hepatic concentrations of vitamin A and folate, elevated vitamin E concentration in serum and substantial *To whom correspondence should be addressed. tPresent address: Nutrition International, Inc., East Brunswick, NJ 08816, USA. Abbreviations: BrdU = bromodeoxyuridine; HCT = haematocrit; HGB = haemoglobin: NaSac = sodium saccharin; NMR = nuclear magnetic resonance; RBC = red blood cells; SEM = scanning electron microscopy. increases in serum lipid concentrations (Garland et al., 1991a). Most of these effects were dose dependent and were reversible by 90 days of age, even with continued NaSac administration (Garland et al., 1991b). Some of the nutritional and biochemical effects of NaSac treatment in young rats were similar to those found in pups of iron-deficient dams (O'Connor et al., 1989; Rao and Larkin, 1988 and 1989; Sherman, 1979; Sherman et al., 1978 and 1982). For example, the offspring of iron-deficient dams had decreased body weights, were anaemic, exhibited increased serum cholesterol and triglyceride levels and were folate-deficient, in comparison with the offspring of control dams. Based on these similarities, the present experiment was conducted to determine whether the effects of 7.5% dietary NaSac could be counteracted by iron and/or folate supplementation. In addition, because the results of previous studies suggested that changes in the urinary milieu might be involved in the patbogenesis of the bladder induced by high dietary levels of NaSac (Ellwein and Cohen, 1990), we evaluated the relationship between the previously mentioned physiological effects and se- lected urinary parameters as well as proliferative lesions in the urinary bladder. 689