Calcif Tissue Int (1993) 53:180-186 Calcified Tissue International © 1993 Springer-Verlag New York Inc. Molybdenum-Induced Changes in the Epiphyseal Growth Plate Nicola M. A. Parry, 1 Michael Phillippo, 1 Martin D. Reid, 1 Brian A. MeGaw, 1 David J. Flint, z and Nigei Loveridge 1 1Division of Biochemical Sciences, Rowett Research Institute, Greenburn Road, Aberdeen AB2 9SB; and ZHannah Research Institute, Ayr KA6 5HL Scotland Received November 4, 1992; and in revised form January 8, 1993. Summary. Molybdenum (Mo), at high concentrations, in- duces changes in the epiphyseal growth plate through its effects on copper (Cu) metabolism but it is unclear whether or not Mo can induce changes independent of its effects on copper status. To this end, the effect of Mo on longitudinal bone growth was examined in rats. Dietary Mo was given either as ammonium heptamolybdate or as ammonium tetrathiomolybdate, the latter producing a marked Cu defi- ciency. There was a significant reduction in longitudinal bone growth in both groups; however, growth plate width was increased only in the Cu-deficient animals due to an increase in the width of the zone of transitional/hypertrophic chondrocytes. Both glucose 6-phosphate dehydrogenase ac- tivity and cell proliferation (assessed by bromodeoxyuridine incorporation) were markedly decreased in the proliferating zone of the growth plate in both Mo-treated groups. These changes were not apparently related to changes in circulating vitamin D metabolites or insulin-like growth factor-1. The results indicate that excess Mo impairs cell proliferation within the growth plate, whereas the effects of copper defi- ciency are more related to chondrocyte differentiation. Thus, Mo can induce changes in longitudinal bone growth which are distinct from those resulting from Cu deficiency. Key words: Growth plate - Molybdenum - Cell proliferation - Cell differentiation - Chondrocytes. Certain trace elements such as zinc, manganese, and cooper are essential for normal growth and development [1, 2] and there is increasing evidence that other trace elements such as lead and cadmium affect the skeleton [3, 4] and may play a role in the etiology of osteoporosis [5, 6]. Skeletal abnormal- ities due to dietary copper (Cu) deficiency have been ob- served in many species. Copper deficiency results in thin- ning of the cortex and a widened epiphyseal growth plate, along with irregular bone matrix deposition and osteoporosis associated with reduced osteoblastic activity. These findings have been consistently reported in pigs [7-9], chickens [10], and sheep [11]. Similar morphological changes in bone have also been reported in growing rats fed a diet supplemented with high concentrations of molybdenum (Mo) (100-500 I~g/g) [12]. However, bone lesions have been induced in cattle [13] and sheep [14] with Mo concentrations (5-20 Ixg/g), probably due to the interaction between dietary Mo and sulfur in the rumen environment to form thiomolybdate, a potent antag- Offprint requests to: N. Loveridge onist of Cu metabolism [15, 16]. Whereas monogastric ani- mals can tolerate relatively high levels of Mo, the inclusion of dietary Mo in the form of tetrathiomolybdate (6-20 I~g/g) induces bone lesions [17, 18]. There is, however, increasing uncertainty as to whether all the effects of Mo are due to Cu deficiency or Mo [19]. The aim of this study was to determine the effects of Mo on the epiphyseal growth plate and to distinguish these ef- fects from those of Cu deficiency. Materials and Methods Animals and Diets Male Hooded Lister rats of the Rowett strain were weaned at 19 days onto a stock diet for 10 days, followed by ad libitum access to a semisynthetic, albumin-based trace element diet [20, 21]. Animals were then group-housed and randomized according to weight into three different dietary groups. The control group received a basal diet containing 3 mg Cu/kg without any added Mo; the other groups received the basal diet supplemented with 6 mg Mo/kg either as ammonium tetrathiomolybdate (TTM), prepared as described in [22] or ammonium heptamolybdate (AM) (BDH, Poole, Dorset, UK). This concentration of TTM was chosen because a previous study [23] had indicated that this dose induced gross bone lesions but was not generally toxic. Diets were fed for 26 days, with six rats from each group being killed after 13 days, and a further six after 26 days. Mean body weight in the TTM group was reduced slightly at 13 days (control 160.8 -+ 4.35; TTM 144.3 +- 5.28; AM 158.0 +- 1.32; mean +- SEM) but markedly reduced at 26 days (control 262.0 + 3.51; TTM 200.5 -+ 6.03; AM 260.2 -+ 6.24). Tissue Collection and Preparation Animals were injected intraperitoneally with 25 mg 5-bromo- 2'deoxyuridine (BrdUrd) (Sigma) per kg body weight 1 hour before death. Blood was taken into heparinized vacutainer tubes after car- diac puncture under barbiturate anesthesia, centrifuged at 4°C, and the plasma stored at -20°C. Right tibiae were briefly immersed in 5% polyvinyl alcohol (PVA) and chilled to - 70°C in hexane prior to mounting in 5% PVA [24] for cutting serial sections of 10 p, thick- ness on a cryostat (Brights Instruments, Huntingdon, England). Biochemical Analyses Four hundred microliters of 10% trichloroacetic acid (TCA) solution was added to 100 i~1 of plasma and centrifuged for 15 minutes at 3,000 rpm, and the acid-soluble or "available" Cu fraction was an- alyzed on a Pye Unicam SP9 atomic absorption spectrophotometer. Freeze-dried liver samples were microwave acid-digested prior to determination of Cu, Mo, iron (Fe), and zinc (Zn) concentrations by