Changes in gaseous concentration of alkylpyrazine analogs affect mouse avoidance behavior.

We developed a rapid and accurate method for quantifying gaseous phase odorants using headspace solid-phase microextraction (HS-SPME) in conjunction with GC-MS and used our system to quantify alkylpyrazine analogs in the Y-maze. Rapid extraction of volatile compounds in the vapor phase achieved accurate quantitative analysis of gaseous alkylpyrazine analogs at several locations in the Y-maze. We also used a series of three SPME fibers to quantify changes in the concentration over time. We conducted a behavioral test of mice in response to these alkylpyrazines and identified a positive relationship between the rate of increase in gaseous concentration and the avoidance rate induced. Our results demonstrate that the Y-maze is a simple but reliable apparatus for behavioral studies of olfaction. The HS-SPME fast extraction method can quantify how gaseous concentrations of alkylpyrazines change over time, and the time-dependent increase of alkylpyrazine concentration is correlated with induction of aversive behavior in mice.

Application of integrative physiological approach to evaluate human physiological responses to the inhalation of essential oils of Japanese citrus fruits iyokan (Citrus iyo) and yuzu (Citrus junos).

This study investigated the effects of essential oil odors from Japanese citrus fruits, iyokan (Citrus iyo) and yuzu (Citrus junos), on human psychology and both the autonomic and central nervous systems. The inhalation of both essential oils significantly increased miosis rate and fingertip temperature and could induce parasympathetic dominance by suppressing sympathetic nerve activity. Oxyhemoglobin concentration in the prefrontal cortex increased after the inhalation of yuzu essential oil and decreased after the inhalation of iyokan essential oil. Subjectively, the inhalation of both essential oils reduced the feelings of fatigue and improved the feelings of refreshment, suggesting that the effect of autonomic nervous activity might involve in these psychological changes directly. Moreover, we observed that task performance improved after inhaling yuzu essential oil, which may be due to the increase in oxyhemoglobin concentration in the prefrontal cortex.

Fucoxanthin administration delays occurrence of tumors in xenograft mice by colonospheres, with an anti-tumor predictor of glycine.

Fucoxanthin and its major metabolite, fucoxanthinol, have potent anti-cancer properties in carcinogenic model mice and against cancer cells. Evidence has accumulated regarding the diagnostic potential of biological metabolites as invasive and non-invasive obtainable approaches. We recently demonstrated that glycine was an effective predictor of the suppression of sphere formation and epithelial mesenchymal transition by fucoxanthinol in human colorectal cancer stem-like spheroids (colonospheres) under normoxia and hypoxia. In the present study, we investigated the suppressive effect of fucoxanthin on tumorigenesis derived from colonospheres in xenograft mice, and the alteration on the metabolite profiles of mouse tumors by fucoxanthin was evaluated. Fucoxanthin administration at 2.5 mg/kg body weight (p.o.) for 4 weeks significantly inhibited the incidence of tumors by inoculation of colonospheres suspension in BALB/c nu/nu mice compared with control mice, but not tumor sizes. In addition, fucoxanthin down-regulated tumor Cyclin D1 expression by 0.7-fold of that observed in the tumors of the control mice. Moreover, the tumor glycine level in the xenograft mice was decreased by fucoxanthin administration to 0.5-fold. These results imply the possibility of tumor metabolites as a prediction marker of tumorigenicity derived from colorectal cancer stem cells in mice.

Structure-Activity Relationships of Alkylpyrazine Analogs and Fear-Associated Behaviors in Mice.

Our previous studies identified alkyl pyrazine analogs in wolf urine that act as novel kairomones and induce a series of fear-associated behaviors in mice. A mixture of these alkyl pyrazines also effectively suppressed the approach of deer to a feeding area, and animals that did approach the marked area exhibited fear-associated behaviors. To investigate structure-activity relationships of alkyl pyrazines, four fear-associated behaviors - freezing, locomotion activity, odor investigation, and avoidance - were measured in experiments on female C57BL/6 J mice. Of the 17 compounds tested, 2,3-diethylpyrazine, 3-ethyl-2,5-dimethylpyrazine, and 3-ethyl-2,5-dimethylpyrazine induced all four fear-associated behaviors. 2,3,5-Trimethylpyrazine also induced three of the fear-associated behaviors, but did not decrease locomotion. Multivalent analysis of behaviors clearly demonstrated that these four compounds formed an independent cluster and were the most active. Structure-activity relationships revealed that active alkyl pyrazines inducing all four fear-associated behaviors had methyl or ethyl group(s), but not longer carbon chains, and alkyl side chains consisting of four carbon atoms in total were present in the most potent analogs. This study is the first experimental investigation of structure-activity relationships between alkyl pyrazine analogs and fear-associated behaviors in mice.

Evaluation of the masking of dimethyl sulfide odors by citronellal, limonene and citral through the use of trained odor sensor mice.

Previous studies indicate that the most common result of mixing two odors is the decreased olfactory perception of one or both components in the mixture. An excellent example of this phenomenon is provided by the masking of an unpleasant odor by a pleasant odor. This study hypothesized that dimethyl sulfide (DS; a major chemical component of oral malodor) might be masked by citronellal, a monoterpene aldehyde that produces an intense lemon aroma. To investigate this hypothesis, mice were chosen as odor sensor animals. Mice were trained in a Y maze to discriminate between DS (10 ppm in aqueous solution) and water. A series of generalization tests revealed that these trained "odor sensor" mice could also distinguish between 1 ppm DS and water, but not between 0.1 ppm DS and water. The mice were then confronted with the original choice of 10 ppm DS and water, but now in the presence of various concentrations of citronellal. The mice failed to detect the odor of DS when confronted with 30 ppm citronellal. The mice were similarly confused when confronted with citral (90 ppm) or limonene (3000 ppm). This study is the first to show that citrus odorants can mask the odor of DS, altering the behavioral responses of trained odor sensor mice.

Citronellal ingestion decreases the appeal of male mouse urinary pheromone for female mice.

To determine whether ingestion of citronellal decreases the attractive power of the male mouse urinary odor, female mice were used in preference tests. A series of tests revealed that the female mice preferred voided urine odors from aged mice over those from younger adult mice. However, exogenous citronellal directly inhibited the advantage of the aged males with regard to attraction.

Repeated exposure to kairomone-containing coffee odor improves abnormal olfactory behaviors in heterozygous oxytocin receptor knock-in mice.

The oxytocin receptor (OXTR) knockout mouse is a model of autism spectrum disorder, characterized by abnormalities in social and olfactory behaviors and learning. Previously, we demonstrated that OXTR plays a crucial role in regulating aversive olfactory behavior to butyric acid odor. In this study, we attempted to determine whether coffee aroma affects the abnormal olfactory behavior of OXTR-Venus knock-in heterozygous mice [heterozygous OXTR (±) mice] using a set of behavioral and molecular experiments. Four-week repeated exposures of heterozygous OXTR (±) mice to coffee odor, containing three kairomone alkylpyrazines, rescued the abnormal olfactory behaviors compared with non-exposed wild-type or heterozygous OXTR (±) mice. Increased Oxtr mRNA expression in the olfactory bulb and amygdala coincided with the rescue of abnormal olfactory behaviors. In addition, despite containing the kairomone compounds, both the wild-type and heterozygous OXTR (±) mice exhibited a preference for the coffee odor and exhibited no stress-like increase in the corticotropin-releasing hormone, instead of a kairomone-associated avoidance response. The repeated exposures to the coffee odor did not change oxytocin and estrogen synthetase/receptors as a regulator of the gonadotropic hormone. These data suggest that the rescue of abnormal olfactory behaviors in heterozygous OXTR (±) mice is due to the coffee odor exposure-induced OXTR expression.

Alteration of mouse urinary odor by ingestion of the xenobiotic monoterpene citronellal.

Body odors provide a rich source of sensory information for other animals. There is considerable evidence to suggest that short-term fluctuations in body odor can be caused by diet; however, few, if any, previous studies have demonstrated that specific compounds can directly mask or alter mouse urinary odor when ingested and thus alter another animal's behavior. To investigate whether the ingestion of citronellal, a monoterpene aldehyde that produces an intense aroma detected by both humans and mice, can alter mouse urinary odor, mice (C57BL6J) were trained in a Y maze to discriminate between the urinary odors of male donor mice that had ingested either citronellal in aqueous solution or a control solution. Trained mice could discriminate between urinary odors from the citronellal ingestion and control groups. A series of generalization tests revealed that citronellal ingestion directly altered mouse urinary odor. Moreover, trained mice that had successfully discriminated between urinary odors from donor mice of different ages failed to detect age-related changes in urine from male mice that had ingested 50 ppm of citronellal. This study is the first to show that ingestion of a xenobiotic can alter mouse urinary odor and confuse the behavioral responses of trained mice to age-related scents.

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.

Profiles of volatiles in male rat urine: the effect of puberty on the female attraction.

Rat urine contains many volatile constituents that may be used for chemical communication. The levels of certain urinary volatiles are strongly dependent on the sex and endocrine status (e.g., puberty). We performed chemical and behavioral studies to identify the volatiles in adult male rat urine that attract mature females. Our results demonstrated that adult male rats have higher levels of 2-heptanone (2-HP), 4-methylphenol (4-MP), and 4-ethylphenol (4-EP) than prepubescent male rats; furthermore, female rats are more attracted to the odor of adult male rat urine than that of prepubescent males. When prepubescent rat urine was supplemented with 2-HP, 4-MP, and 4-EP to the levels found in adult male urine, the attractiveness of the urine to females was markedly enhanced. Our results suggested that this attraction is due to an increased level of chemosignaling.

The identification of attractive volatiles in aged male mouse urine.

In many species, older males are often preferred mates because they carry "good" genes that account for their viability. In some animals, including mice, which rely heavily on chemical communication, there is some indication that an animal's age can be determined by its scent. In order to identify the attractants in aged male mouse urine, chemical and behavioral studies were performed. We herein show that aged mice have higher levels of 3,4-dehydro-exo- brevicomin (DB), 2-sec-butyl-4,5-dihydrothiazole (BT), and 2-isopropyl-4,5-dihydrothiazole (IT) and a lower level of 6-hydroxy-6-methyl-3-heptanone relative to adult male mice. We also demonstrate that the attraction of females to the odor of male mouse urine is greater when the urine is from aged males. However, the attraction of aged urine odor was offset by the ultrafiltration of adult and aged mouse urine. When DB, BT, and IT were added to adult urine, the attraction of the urine was enhanced. Our results suggest that inbred aged male mice develop an aging odor that is attractive to female mice in an experimental setting and that this attraction is due to increased mouse pheromone signaling.

Syntheses of 2-Isopropyl-4,5-dihydrothiazole and 6-Hydroxy-6-methyl-3-heptanone, pheromone components of the male mouse, Mus musculus.

Syntheses of 2-isopropyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, pheromone components of the male mouse, Mus musculus, were achieved to provide sufficient amounts of samples for biological studies.

Effect of an orally ingested mugwort and mushroom extract mixture on urine odor from aged mice.

We had previously found that male mice could be trained to discriminate between the urine odor of aged and young adult (adult) mice. We hypothesized that these odors that characterized the older animals might be inhibited by a mixture of extracts (AAM) of mugwort and mushroom, because previous studies have indicated that these extracts could be used to reduce the intensity of unpleasant body odors. The findings of this chemical study strongly suggest that the AAM function helped to modify the aged mouse urine odor so that it more closely resembled the smell of urine from younger mice. Based on the results of the chemical studies, a set of behavioral experiments were therefore conducted. The results of three sets of generalization trials also strongly supported the results of the chemical studies. Together, these results suggest that ingested AAM decreased the intensity of odors associated with aging in mice.

Oxytocin receptor signaling contributes to olfactory avoidance behavior induced by an unpleasant odorant.

Oxytocin (OXT) and its receptor (OXTR) regulate reproductive physiology (i.e. parturition and lactation), sociosexual behavior, learned patterns of behavior and olfactory behavior in social contexts. To characterize the function of OXTR in basic olfactory behavior, the present study compared the behavioral responses of homozygous, heterozygous and wild-type mice when these mice were confronted with an unpleasant odorant (butyric acid) in a custom-made Y-maze in the absence of a social context. Wild-type mice avoided the first encounter with the butyric acid odorant, whereas homozygous and heterozygous mice did not. However, both heterozygous and wild-type mice habituated when confronted with the butyric odorant again on the following 2 days. By contrast, homozygous mice failed to habituate and instead avoided the location of the odorant for at least 3 days. These data suggest that homozygous and heterozygous mice display abnormal olfactory responses to the presentation of an unpleasant odorant. Our studies demonstrate that OXTR plays a critical role in regulating olfactory behavior in the absence of a social context.

Glycine and succinic acid are effective indicators of the suppression of epithelial-mesenchymal transition by fucoxanthinol in colorectal cancer stem-like cells.

Fucoxanthinol (FxOH) is a strong anticancer metabolite of fucoxanthin that accumulates in abundance in edible brown algae and promises human health benefits. FxOH has been shown to suppress tumorigenicity and sphere formation in human colorectal cancer stem cell (CCSC)-like spheroids (colonospheres, Csps). In the present study, we aimed to clarify the inhibitory activity of FxOH on epithelial-mesenchymal transition (EMT), which is essential for cancer recurrence and distant metastasis, and to identify intracellular low-molecular-weight metabolites that may be useful for evaluating the cellular effects of FxOH on CCSCs. FxOH significantly suppressed sphere-forming activity, migration and invasion in a dose-dependent manner. In addition, treatment with 50 µmol/l FxOH suppressed N-cadherin and vimentin expression and the activation of integrin signaling linked to EMT suppression by western blot analysis. MAPK signaling and STAT signaling related to cell growth and apoptosis in Csps derived from human CRC HT-29 and HCT116 cells were also altered. According to our metabolite profiling by GC-MS analysis, reduced glycine and succinic acid levels were correlated with EMT suppression and apoptosis induction in Csps. Our data indicate that simple amino acids such as glycine and succinic acid may be good prognostic indicators of physiological changes to CCSCs induced by FxOH treatment.

Glycine Is a Predictor for a Suppressive Effect of Fucoxanthinol on Colonosphere Formation Under Hypoxia.

BACKGROUND/AIM: Fucoxanthinol (FxOH), a metabolite of fucoxanthin, is known to inhibit tumorigenicity of human colorectal cancer stem cells (CCSCs) and their sphere formation. Hypoxic conditions and hypoxia-inducible factors (HIFs) are essential to maintain the stemness of CCSCs. We investigated effects of FxOH on sphere formation, intercellular energy metabolites in colonospheres formed from human colorectal HT-29 cells under hypoxic conditions. RESULTS: FxOH at 50 µM suppressed HIF1α expression and activation of integrin, mitogen-activated protein kinase (MAPK), Wingless/integrated (WNT) and signal transducer and activator of transcription (STAT) signals. Moreover, expression of epithelial-mesenchymal transition- and apoptosis-related proteins in the colonospheres was lowered by FxOH. The level of glycine was reduced in hypoxic colonospheres under FxOH treatment. CONCLUSION: FxOH attenuated the sphere formation of hypoxic colonospheres, in part, by suppressing HIF1α expression. Glycine could be a potential predictor for the activity of agents that inhibit sphere formation by hypoxic colonospheres.

Etizolam attenuates the reduction in cutaneous temperature induced in mice by exposure to synthetic predator odor.

Anxiety- and stress-related disorders can be debilitating psychiatric conditions in humans. To prevent or ameliorate these conditions, reliable animal models are needed to evaluate the effects of anxiolytic drugs. Previously, we found that a mixture of three pyrazine analogues (P-mix) that were present at high levels in wolf urine induced fear-related responses in mice, rats and deer. A change in cutaneous temperature was shown to be induced by acute stress simultaneously with changes in heart rate, arterial pressure and freezing behavior, raising the possibility that cutaneous temperature could be used as an index of stress. In the present study, using infrared thermography, we showed that exposure of mice to P-mix induced a decrease in cutaneous temperature. We then examined the dose-dependent effects of an anxiolytic drug, etizolam (0-20 mg/kg), on the temperature decrease. Pre-administration of etizolam (5 mg/kg or higher) inhibited the P-mix-induced decrease in cutaneous temperature. Exposure to P-mix induced Fos-immunoreactivity, a marker of neuronal excitation, at the mouse amygdala and hypothalamus, and etizolam (5 mg/kg) attenuated that immunoreactivity. The present results suggested that the measurement of cutaneous P-mix-induced temperature changes in mice could be used as an animal model for evaluating the effects of anxiolytic drugs.

Pyrazine analogues from wolf urine induced unlearned fear in rats.

Urine excreted from the common grey wolf (Canis lupus) contains a kairomone, inducing fear-related behaviors in various mammals. Numerous fear-inducing substances activate neurons at the main and/or accessory olfactory bulb (AOB), medial and central amygdala, and hypothalamus. Our previous study showed that the mixture of pyrazine analogues (P-mix) contained in wolf urine induced avoidance and fear-related behaviors in laboratory mice and Hokkaido deer (Cervus nippon yesoensis), a species native to Japan. Exposure to wolf urine or P-mix induced expression of Fos, a marker of neuronal excitation, in the AOB of mice. In the present study, we explored the effects of P-mix on fear-related behaviors and Fos-expression in rats. Exposure to P-mix induced avoidance and immobilization in rats, while that to a mixture of i-amyl acetate, linalool and R(+)-limonene (O-mix), which generate floral and fruity odors, induced avoidance but not immobilization. P-mix but not O-mix increased Fos-immunoreactivity of the AOB, medial and central amygdala, and hypothalamus of rats. The present results suggest that P-mix odor induces unlearned fear-related behaviors in rats.

Olfactory fingerprints for major histocompatibility complex-determined body odors II: relationship among odor maps, genetics, odor composition, and behavior.

The olfactory system detects small differences in the composition of natural odorants, made up of hundreds of molecules. Odorous quality is hypothetically represented by a combinatorial code: activation of distinct but overlapping subsets of olfactory receptors resulting in activation of a distinct subset of glomeruli in the main olfactory bulb (MOB). Here we show that modification of a single gene (the K gene of the major histocompatibility locus), which results in a subtle change in the odiferous quality of urine, causes a small but significant change in the composition of urine volatiles and consequently the evoked glomerular activation pattern in the MOB. The magnitude of disparity between urine-evoked glomerular activation patterns is predictive of the extent of (1) the genetic difference among the urine donors, (2) the difference in the chemical composition of urine, and (3) the odor detector's ability to discriminate. These data on natural odors are consistent with the combinatorial code hypothesis and identify subsets of glomeruli that are apt to play a significant role in mediating individual recognition.

The scent of wolves: pyrazine analogs induce avoidance and vigilance behaviors in prey.

The common gray wolf (Canis lupus) is an apex predator located at the top of the food chain in the Northern Hemisphere. It preys on rodents, rabbits, ungulates, and many other kinds of mammal. However, the behavioral evidence for, and the chemical basis of, the fear-inducing impact of wolf urine on prey are unclear. Recently, the pyrazine analogs 2, 6-dimethylpyrazine, 2, 3, 5-trimethylpyrazine and 3-ethyl-2, 5-dimethyl pyrazine were identified as kairomones in the urine of wolves. When mice were confronted with a mixture of purified pyrazine analogs, vigilance behaviors, including freezing and excitation of neurons at the accessory olfactory bulb, were markedly increased. Additionally, the odor of the pyrazine cocktail effectively suppressed the approach of deer to a feeding area, and for those close to the feeding area elicited fear-related behaviors such as the "tail-flag," "flight," and "jump" actions. In this review, we discuss the transfer of chemical information from wolf to prey through the novel kairomones identified in wolf urine and also compare the characteristics of wolf kairomones with other predator-produced kairomones that affect rodents.