Butylated hydroxytoluene is a ligand of urinary proteins derived from female mice.

Mice secrete substantial amounts of protein, particularly proteins called the major urinary proteins (MUPs), in urine. One function of MUPs is to sequester volatile pheromone ligands, thereby delaying their release and providing a stable long-lasting signal. Previously, only MUPs isolated from male mice have been used to identify ligands. Here, we tested the hypothesis that MUPs derived from females may also sequester volatile organic compounds. We identified butylated hydroxytoluene (BHT), a synthetic antioxidant present in the laboratory rodent diet, as a major ligand bound to urinary proteins derived from C57BL/6J female urine. BHT was also bound to the male-derived proteins, but the binding was less prominent than that in female urine, even though males express approximately 4 times more proteins than females. We confirmed that the majority of BHT in female urine was associated with the high molecular weight fraction (>10 kDa) and the majority of the proteins that sequestered BHT were MUPs as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequestration of BHT by MUPs was further confirmed by employing the recombinant MUP8 whose natural analogue has been reported in both sexes. Therefore, our data indicate that MUPs expressed in both sexes can bind, transport, and excrete xenobiotics into urine and raise the possibility that in addition to the known role in chemical communication, MUPs function as a defense mechanism against exogenous toxins.

Major histocompatibility complex-regulated odortypes: peptide-free urinary volatile signals.

Major histocompatibility complex (MHC) genes influence urinary odors (odortypes) of mice. That volatile odorants are involved is supported by the observation that odortype identity can be detected from a distance. Furthermore, chemical analyses of urines have revealed numerous volatile odorants that differ in relative abundance between mice that differ only in MHC genotypes. In addition, urines from MHC-different mice evoke distinct odor-induced activity maps in the main olfactory bulbs. However, recent studies report that non-volatile MHC class I peptides may directly act as MHC-associated signals and may thereby be seen to call into question the evidence for a volatile MHC signal. To evaluate this question, we designed a procedure to collect peptide-free urinary volatiles and tested these volatiles for their ability to mediate chemosensory discrimination of MHC-congenic mice differing in their MHC genotype. The headspace volatiles from urines of C57BL/6 congenic mice (haplotypes H2(b) and H2(k)) were collected by solid phase microextraction (SPME). These volatiles were then desorbed into a gas chromatograph (GC) and the entire chromatographic eluate was collected into a buffer solution. Our results conclusively demonstrate that mice trained to discriminate between unadulterated urinary signals of the congenic mice generalize the discrimination, without reward or training, to the buffer solution containing the peptide-free urinary volatiles (p<0.001, binomial test). Thus volatile signals, perhaps along with non-volatile ones, are capable of mediating behavioral discriminations of mice of different MHC genotypes.