2-sec-butyl-4,5-Dihydrothiazole Is a Ligand for Mouse Urinary Protein and Rat α2u-Globulin: Physiological and Toxicological Relevance

Abstract

Mouse urinary protein (MUP) and α2u-globulin are structurally homologous proteins that belong to a superfamily of ligand-binding proteins and represent the major urinary proteins excreted by adult male mice and rats, respectively. Although a variety of xenobiotics bind to α2u-globulin and produce a male rat-specific hyaline droplet nephropathy, no endogenous ligand for this protein has been identified. Despite extensive sequence homology, MUP does not bind to hyaline droplet-inducing agents. While performing experiments with purified MUP, we observed that it presented with a strong, distinctive odor reminiscent of mouse urine. To determine whether this odor was the result of contamination or degradation or was attributed to an endogenous ligand bound to the protein, the protein was subjected to thermal desorption and any released volatile compounds were detected with a gas chromatograph equipped with an external sniff port and mass spectrometer. With this approach, two odorous compounds were detected at the sniff port by a human observer, but only one was present in sufficient mass to allow identification. This compound, which presented with the characteristic odor, was subsequently identified as 2-secbutyl-4,5-dihydrothiazole (DHT) by GC/MS/matrix isolation IR and NMR analyses. The identification of DHT was confirmed by comparing the chromatographic and spectral properties to those of the synthesized authentic compound. In direct contrast, purified urinary α2u-globulin did not present with an obvious odor, and no volatile ligands were detected on this protein. Although DHT is a major endogenous ligand for MUP, it was also found to competitively inhibit the binding of [14C]d-limonene-1,2-epoxide to α2u-globulin with relatively high affinity (Ki= 2.3 μM). When dosed orally to F344 rats, DHT (1 mmol/kg for 3 days) caused the characteristic exacerbation of hyaline droplets in male rat kidneys and increased renal levels of immunoreactive α2u-globulin about threefold over control levels. These results indicate that despite structural homology, MUP and α2u-globulin are distinguished by the presence of a volatile endogenous ligand only on the former, a distinction that may reflect differences in the physiological functions of the two proteins. Furthermore, although DHT can bind to both MUP and α2u-globulin, renal toxicity was only observed in rats, thereby emphasizing the unique toxicological properties of α2u-globulin in the development of hyaline droplet nephropathy.