We present here a novel integrative metabonomic approach to probe toxic effects of drugs in experimental animals using alpha-naphthylisothiocyanate (ANIT) as a model hepatotoxicant. Male Han-Wistar rats were dosed with ANIT (150 mg/kg, n = 25), and plasma and liver samples were collected for NMR and magic-angle spinning (MAS) NMR spectroscopy at 3, 7, 24, 31, and 168 h postdosing. Urine was collected continuously for 3 days prior to dosing and up to 168 h postdose. Histopathology and plasma clinical chemistry was also performed at all time points. Liver samples were analyzed either intact by 600 MHz 1H MAS NMR techniques or using high resolution (liquid state) 1H NMR of water-acetonitrile extracts. These data were related to sequential 1H NMR measurements in urine and plasma using pattern recognition methods. 1D 1H NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by ANIT toxicity. From the eigenvector loadings of the PCA, those regions of the 1H NMR spectra and hence the combinations of endogenous metabolites marking the main phase of the toxic episode were identified. The ANIT-induced biochemical manifestations included a hepatic lipidosis associated with hyperlipidaemia; hyperglycaemia and glycosuria; increased urinary excretion of taurine and creatine; a shift in energy metabolism characterized by increased plasma ketone bodies with reduced urinary excretion of tricarboxylic acid cycle intermediates and raised hepatic bile acids leading to bile aciduria. The integration of metabolic data derived from several sources gives a holistic approach to the study of time-related toxic effects in the intact system and enables the characterization of key metabolic effects during the development and recovery from a toxic lesion.

By proposing TSH as a key negative regulator of bone turnover, recent studies in TSH receptor (TSHR) null mice challenged the established view that skeletal responses to disruption of the hypothalamic-pituitary-thyroid axis result from altered thyroid hormone (T(3)) action in bone. Importantly, this hypothesis does not explain the increased risk of osteoporosis in Graves' disease patients, in which circulating TSHR-stimulating antibodies are pathognomonic. To determine the relative importance of T(3) and TSH in bone, we compared the skeletal phenotypes of two mouse models of congenital hypothyroidism in which the normal reciprocal relationship between thyroid hormones and TSH was intact or disrupted. Pax8 null (Pax8(-/-)) mice have a 1900-fold increase in TSH and a normal TSHR, whereas hyt/hyt mice have a 2300-fold elevation of TSH but a nonfunctional TSHR. We reasoned these mice must display opposing skeletal phenotypes if TSH has a major role in bone, whereas they would be similar if thyroid hormone actions predominate. Pax8(-/-) and hyt/hyt mice both displayed delayed ossification, reduced cortical bone, a trabecular bone remodeling defect, and reduced bone mineralization, thus indicating that the skeletal abnormalities of congenital hypothyroidism are independent of TSH. Treatment of primary osteoblasts and osteoclasts with TSH or a TSHR-stimulating antibody failed to induce a cAMP response. Furthermore, TSH did not affect the differentiation or function of osteoblasts or osteoclasts in vitro. These data indicate the hypothalamic-pituitary-thyroid axis regulates skeletal development via the actions of T(3).

Euthyroid status is essential for normal skeletal development and maintenance of the adult skeleton, but the mechanisms which control supply of thyroid hormone to bone cells are poorly understood. Thyroid hormones enter target cells via monocarboxylate transporter-8 (MCT8), which provides a functional link between thyroid hormone uptake and metabolism in the regulation of T3-action but has not been investigated in bone. Most circulating active thyroid hormone (T3) is derived from outer ring deiodination of thyroxine (T4) mediated by the type 1 deiodinase enzyme (D1). The D2 isozyme regulates intra-cellular T3 supply and determines saturation of the nuclear T3-receptor (TR), whereas a third enzyme (D3) inactivates T4 and T3 to prevent hormone availability and reduce TR-saturation. The aim of this study was to determine whether MCT8 is expressed in the skeleton and whether chondrocytes, osteoblasts and osteoclasts express functional deiodinases. Gene expression was analyzed by RT-PCR and D1, D2 and D3 function by sensitive and highly specific determination of enzyme activities. MCT8 mRNA was expressed in chondrocytes, osteoblasts and osteoclasts at all stages of cell differentiation. D1 activity was undetectable in all cell types, D2 activity was only present in mature osteoblasts whereas D3 activity was evident throughout chondrocyte, osteoblast and osteoclast differentiation in primary cell cultures. These data suggest that T3 availability especially during skeletal development may be limited by D3-mediated catabolism rather than by MCT8 mediated cellular uptake or D2-dependent T3 production.