Urinary Volatile Compounds as Biomarkers for Lung Cancer: A Proof of Principle Study Using Odor Signatures in Mouse Models of Lung Cancer

A potential strategy for diagnosing lung cancer, the leading cause of cancer-related death, is to identify metabolic signatures (biomarkers) of the disease. Although data supports the hypothesis that volatile compounds can be detected in the breath of lung cancer patients by the sense of smell or through bioanalytical techniques, analysis of breath samples is cumbersome and technically challenging, thus limiting its applicability. The hypothesis explored here is that variations in small molecular weight volatile organic compounds (“odorants”) in urine could be used as biomarkers for lung cancer. To demonstrate the presence and chemical structures of volatile biomarkers, we studied mouse olfactory-guided behavior and metabolomics of volatile constituents of urine. Sensor mice could be trained to discriminate between odors of mice with and without experimental tumors demonstrating that volatile odorants are sufficient to identify tumor-bearing mice. Consistent with this result, chemical analyses of urinary volatiles demonstrated that the amounts of several compounds were dramatically different between tumor and control mice. Using principal component analysis and supervised machine-learning, we accurately discriminated between tumor and control groups, a result that was cross validated with novel test groups. Although there were shared differences between experimental and control animals in the two tumor models, we also found chemical differences between these models, demonstrating tumor-based specificity. The success of these studies provides a novel proof-of-principle demonstration of lung tumor diagnosis through urinary volatile odorants. This work should provide an impetus for similar searches for volatile diagnostic biomarkers in the urine of human lung cancer patients.     

In search of the chemical basis for MHC odourtypes

Mice can discriminate between chemosignals of individuals based solely on genetic differences confined to the major histocompatibility complex (MHC). Two different sets of compounds have been suggested: volatile compounds and non-volatile peptides. Here, we focus on volatiles and review a number of publications that have identified MHC-regulated compounds in inbred laboratory mice. Surprisingly, there is little agreement among different studies as to the identity of these compounds. One recent approach to specifying MHC-regulated compounds is to study volatile urinary profiles in mouse strains with varying MHC types, genetic backgrounds and different diets. An unexpected finding from these studies is that the concentrations of numerous compounds are influenced by interactions among these variables. As a result, only a few compounds can be identified that are consistently regulated by MHC variation alone. Nevertheless, since trained animals are readily able to discriminate the MHC differences, it is apparent that chemical studies are somehow missing important information underlying mouse recognition of MHC odourtypes. To make progress in this area, we propose a focus on the search for behaviourally relevant odourants rather than a random search for volatiles that are regulated by MHC variation. Furthermore, there is a need to consider a ‘combinatorial odour recognition’ code whereby patterns of volatile metabolites (the basis for odours) specify MHC odourtypes.     

Chemical mediation of reciprocal mother–offspring recognition in the Southern Water Skink (Eulamprus heatwolei)

Abstract Kin recognition has been demonstrated to play an important role in the social structure of a wide range of animals. Most studies to date have examined parent–offspring recognition only in species that provide offspring with direct parental care, however, there are several advantages to parent–offspring recognition even in the absence of direct parental care. In this study we investigated reciprocal mother–offspring recognition in the Australian scincid lizard Eulamprus heatwolei, a species that does not show direct parental care. We examined whether neonates could discriminate between their mothers and unrelated females, and whether females could discriminate between their offspring and unrelated neonates, via chemical cues, using retreat site selection experiments. We conducted trials when neonates were 1 and 4 weeks old to investigate whether responses are maintained as neonates age. We found that both neonates and mothers could discriminate between related and unrelated individuals when neonates were 1 week old. Mothers were more likely to take refuge under tiles treated with the odours of their own offspring, while neonates spent less time in areas treated with the odours of unrelated females. At 4 weeks of age, mothers no longer exhibited discriminatory behaviour between their offspring and unrelated neonates, while neonates were more likely to associate with the odour of any female over the odourless control. We hypothesize that reciprocal mother–offspring recognition in E. heatwolei reduces interference competition between mothers and their offspring and also may be important in habitat selection and territory establishment.     

Individual odours and mate recognition in the prairie vole,Microtus ochrogaster

A habituation-discrimination technique was used to demonstrate that male and female prairie voles can discriminate individual differences in the odours of soiled shavings and urine from male and female conspecifics. A second experiment, employing a Y-maze, showed that females significantly preferred the odours of their mate over those of either another mated male or an unmated male. Males preferred their mate's odours to those of other mated females but showed no significant preference between the odours of their mate and those of a virgin female. A third experiment demonstrated that, over a 10-h period, females built nests and/or stayed preferentially on the side of a Y-maze containing their mate's odours. Likewise, males preferentially built nests in their mate's side compared to the side containing odours from a virgin female. However, although the same trend was present when mate odours were paired with odours from another mated female, the preference was not statistically significant. Taken as a whole, these results indicate that mate recognition may be an important of individually distinctive odours in this species.     

Reliability of Transcutaneous Measurement of Renal Function in Various Strains of Conscious Mice

Measuring renal function in laboratory animals using blood and/or urine sampling is not only labor-intensive but puts also a strain on the animal. Several approaches for fluorescence based transcutaneous measurement of the glomerular filtration rate (GFR) in laboratory animals have been developed. They allow the measurement of GFR based on the elimination kinetics of fluorescent exogenous markers. None of the studies dealt with the reproducibility of the measurements in the same animals. Therefore, the reproducibility of a transcutaneous GFR assessment method was investigated using the fluorescent renal marker FITC-Sinistrin in conscious mice in the present study. We performed two transcutaneous GFR measurements within three days in five groups of mice (Balb/c, C57BL/6, SV129, NMRI at 3–4 months of age, and a group of 24 months old C57BL/6). Data were evaluated regarding day-to-day reproducibility as well as intra- and inter-strain variability of GFR and the impact of age on these parameters. No significant differences between the two subsequent GFR measurements were detected. Fastest elimination for FITC-Sinistrin was detected in Balb/c with significant differences to C57BL/6 and SV129 mice. GFR decreased significantly with age in C57BL/6 mice. Evaluation of GFR in cohorts of young and old C57BL/6 mice from the same supplier showed high consistency of GFR values between groups. Our study shows that the investigated technique is a highly reproducible and reliable method for repeated GFR measurements in conscious mice. This gentle method is easily used even in old mice and can be used to monitor the age-related decline in GFR.     

Intestinal uptake of fluorescent microspheres in young and aged mice

Rhodamine B-labeled synthetic latex particles (microspheres), 1.8 micron in diameter, were administered by gavage 5 days per week to young (24 days) and aged (18 months) mice. After 25 days (19 gavages), the particles were assayed in solubilized tissues by depositing them on filters and counting under fluorescence microscopy. Aged mice exhibited significantly more fluorescent particle accumulation in Peyer's patches but significantly less in lungs than young mice. Mesenteric lymph nodes and Peyer's patch-free intestinal segments contained measurable latex, but differences between young and aged animals were not significant. Liver contained only trace amounts of latex, and spleen and kidney were latex free in both young and aged animals. Nonquantitative observations on KOH-glycerol-cleared whole Peyer's patches and slices of liver, lung, and mesenteric lymph node were similar.     

鳄蜥对熟悉和陌生个体气味的辨别

个体辨别对于减少同种争斗以及配偶选择具有重要意义。我们用棉棒粘取鳄蜥(Shinisaurus crocodilurus)尿液作为气味源,以香水作为对照,测定鳄蜥对熟悉个体气味、陌生个体气味以及香水的舔舌次数和舔舌潜伏期,来探讨鳄蜥通过化学信息辨别熟悉和陌生个体的能力。结果显示,不论是雌性还是雄性,对不同个体尿液的舔舌次数均显著高于对香水的,舔舌潜伏期显著短于香水的;尽管雄性对陌生同性个体气味与熟悉同性个体气味的舔舌次数无显著差异,但对前者的舔舌潜伏期显著短于后者;雄性对陌生雌性气味的舔舌次数显著多于熟悉雌性气味的,对前者的舔舌潜伏期显著短于后者;雌性对陌生雄性气味的舔舌潜伏期显著短于对熟悉雄性气味的;雄鳄蜥对陌生雌性气味的舔舌次数显著多于雌鳄蜥对陌生雄性的。结果表明,鳄蜥能辨别同种个体的化学信息,并能通过化学信息来辨别熟悉和陌生个体,推测鳄蜥的这种辨别能力对其领域分配以及繁殖交配有重要作用。     

Individual Odours in Two Chromosomal Species of Blind,Subterranean Mole Rat (Spalax ehrenbergi): Conspecific and Cross-species Discrimination

Mole rats from two chromosomal species (2n = 58 and 2n = 60) of the Spalax ehrenbergi superspecies of Israel were tested to determine whether they were able to discriminate differences in the odour of urine from same-sex individuals of their own and of the other chromosomal species. An habituation-discrimination apparatus was designed for use with these solitary and blind subterranean rodents. Animals habituated to the odour of urine from one individual presented for 10 min at a centre sniffing area in the roof of a 50 cm long Perspex tunnel. The odour of urine from the original donor and from a second individual were presented at two other sniffing areas in the tunnel roof during a 5 min discrimination phase. Significant differences in the time spent investigating the two odours demonstrated successful discrimination between them. The results indicate that male and female mole rats of both species can discriminate between the individual-specific odour cues in urine from pairs of conspecifics and pairs of heterospecific mole rats.     

Increased insensible water loss contributes to aging related dehydration

Dehydration with aging is attributed to decreased urine concentrating ability and thirst. We further investigated by comparing urine concentration and water balance in 3, 18 and 27 month old mice, consuming equal amounts of water. During water restriction, 3 month old mice concentrate their urine sufficiently to maintain water balance (stable weight). 18 month old mice concentrate their urine as well, but still lose weight (negative water balance). 27 month old mice do not concentrate their urine as well and lose even more weight than the 18 month old mice, indicating a larger negative water balance. Negative water balance in older mice is accompanied by increased vasopressin excretion, providing further evidence of dehydration. All 3 groups maintain water balance while consuming only the water in gel food containing 56% water. However, both older groups excrete a smaller volume of urine of higher osmolality, indicating greater extra urinary water loss. Since their feces also contain less water, the excess water lost by the older mice apparently is through other routes, presumably insensible loss through the respiratory tract and skin. The greater insensible water loss occurs at an earlier age (18 months) than decreased urine concentrating ability (27 months). We propose that insensible water loss through skin and respiration increases with age, making a major contribution to aging related dehydration.     

Young female pigs can discriminate individual differences in odours from conspecific urine

The ability to discriminate odour cues from different conspecifics has been demonstrated in a variety of small mammalian species. We used a habituation-dishabituation procedure to investigate whether 10-week-old female pigs,Sus scrofa , are able to discriminate between urinary odours from similar-aged conspecifics that were unfamiliar (not encountered for at least 7 weeks). We also examined whether environmental factors can affect the ease with which urine from different individuals is discriminated. Subjects receiving urine samples from the same unfamiliar individual in two successive 2-min exposures separated by a 15-min interval showed habituation in their investigation response to the urine in the second exposure. This habituation was maintained in a third 2-min exposure, 15 min later, if the urine sample was again from the same individual. However, if the urine sample was from a different unfamiliar individual, there was a dishabituation of the investigation response. This was taken to indicate an ability to discriminate the two samples. These data indicate that young pigs could use urinary cues to discriminate other individuals. Effective individual discrimination should facilitate the formation and maintenance of stable social groupings but could be disrupted if, for example, animals from the same group share common odour cues that mask individually distinctive scents. However, urine samples from individuals living in the same group appeared to be no more difficult to discriminate than those from individuals living in different groups. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Olfactory Discrimination of Individual Scents in the Subterranean Rodent Ctenomys talarum (tuco-tuco)

Biological odours that convey cues regarding individual identity can provide valuable information mediating many aspects of mammalian social relationships such us dominance hierarchies, group memberships, territorial and mating activities. The ability of the subterranean rodent, tuco‐tuco (Ctenomys talarum), to discriminate between soiled shavings, urine and faeces from different individuals was investigated using the habituation–discrimination paradigm. Discrimination by both males and females was tested using scents obtained from same‐ and opposite‐sex individuals. Each test subject was habituated for three consecutive days to odour samples from the same individual; on the fourth day a scent from a novel individual was provided. For all odour sources, animals spent significantly less time investigating the habituation scent over successive trials, indicating that animals perceived the stimulus as familiar. For all stimuli, test subjects spent more time investigating the novel odour, rather than the familiar one. Animals spent more time investigating soiled shavings than urine or faeces. Both males and females discriminated novel from familiar odours in shavings and urine regardless of the gender of the odour donors. In contrast, test animals discriminated between familiar and novel odours in feces only when the fecal donors were of different sex from subjects. Possible territorial and reproductive functions of individual scent discrimination are discussed.     

Mate Choice for Non-siblings in Wild House Mice: Evidence from a Choice Test and a Reproductive Test

Two experiments were designed to test whether wild house mice discriminate between olfactory cues from different kin and, if so, whether given preferences would relate to actual reproductive decisions. Experimental animals were mice born to the offspring of wild-caught house mice. Litter-mates stayed together until 60 d of age and were then housed individually. In a choice test, animals were placed in the middle of an arena with 4 openings which led to small cages containing bedding material from opposite-sex animals of known kinship (full-sib, cousin, unrelated) or clean material. Test animals (11 oestrous females, 11 males tested with oestrous females' bedding, 8 males tested with material from non-oestrous females) preferred conspecific to control bedding. Males tested with oestrous females' bedding significantly preferred unrelated to full-sib odours. In a second experiment, 34 males were each mated simultaneously to 3 females (sister, cousin, unrelated) and these groups were then housed together for 5, 10, and 15 d. Females were checked for litters during the next 20 d. Reproductive rate increased significantly in the 15 d cohabitation group, and significantly more cousin and unrelated females than sisters gave birth to a litter.     

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.     

Aging does not Enhance Experimental Cigarette Smoke-Induced COPD in the Mouse

It has been proposed that the development of COPD is driven by premature aging/premature senescence of lung parenchyma cells. There are data suggesting that old mice develop a greater inflammatory and lower anti-oxidant response after cigarette smoke compared to young mice, but whether these differences actually translate into greater levels of disease is unknown. We exposed C57Bl/6 female mice to daily cigarette smoke for 6 months starting at age 3 months (Ayoung@) or age 12 months (Aold@), with air-exposed controls. There were no differences in measures of airspace size between the two control groups and cigarette smoke induced exactly the same amount of emphysema in young and old. The severity of smoke-induced small airway remodeling using various measures was identical in both groups. Smoke increased numbers of tissue macrophages and neutrophils and levels of 8-hydroxyguanosine, a marker of oxidant damage, but there were no differences between young and old. Gene expression studies using laser capture microdissected airways and parenchyma overall showed a trend to lower levels in older animals and a somewhat lesser response to cigarette smoke in both airways and parenchyma but the differences were usually not marked. Telomere length was greatest in young control mice and was decreased by both smoking and age. The senescence marker p21^Waf1 was equally upregulated by smoke in young and old, but p16^INK4a, another senescence marker, was not upregulated at all. We conclude, in this model, animal age does not affect the development of emphysema and small airway remodeling.     

Age-associated differences in cardiovascular inflammatory gene induction during endotoxic stress

Upon physiological stress, families of stress response genes are activated as natural defense mechanisms. Here, we show that induction of specific inflammatory genes is significantly dysregulated and altered in the heart of aged (24--26-month-old) versus young (4-month-old) mice experimentally challenged with a bacterial endotoxin, lipopolysaccharide (LPS, 1.5 mg/kg of body mass). Whereas the LPS-mediated induction of cardiac mRNA for tumor necrosis factor alpha or inducible nitric-oxide synthase showed no age-associated differences, the induction of interleukin-1 beta (IL-1 beta) and intracellular adhesion molecule-1 was modestly extended with aging, and the induction of IL-6 was significantly prolonged with aging. This age-associated phenomenon occurred gradually from 4 to 17 months of age and became more evident after 23 months of age. The age-associated augmentation of the cardiac IL-6 induction was also dramatic at the protein level. Immunohistochemically, the LPS-induced cardiac IL-6 was localized mainly in the microvascular walls. Aged but not young mice showed a high mortality rate during these experiments. These results demonstrate that endotoxin-mediated induction of specific inflammatory genes in cardiovascular tissues is altered with aging, which may be causally related to the increased susceptibility of aged animals to endotoxic stress.     

Clinical and pathogenic studies on aged polyuria in the IVCS strain of mouse

As aged polyuria is often observed in the IVCS strain of mouse, biochemical and histological studies were undertaken in order to clarify its etiology. Polyuria was observed at 7-8 months of age, and significant increases in water intake and urine volume were noted at 10-11 months of age. IVCS strain mice over one year old showed water intakes and urine volumes about five to six times greater than those in DDI strain mice. The osmolarity of urine excreted from polyuric mice was low compared with DDI strain mice, and elevations of sodium and potassium excretion were observed at an early stage of polyuria. At a more advanced stage of the disease, proteins of low molecular weight were excreted in most animals. Furthermore, depression of kidney response to ADH was recognized soon after onset of polyuria compared with normal IVCS strain mice. Thus, polyuria observed in IVCS strain mice may result from a functional defect of the renal tubules. In addition, significant deposition of amorphous substances, especially in the liver, kidney and spleen, occurred almost in parallel with polyuria. From these findings, it is obvious that mice of the IVCS strain exhibit characteristic polyuria and storage disease as they age.     

Dietary iron restriction increases plaque stability in apolipoprotein-E-deficient mice

Accumulative evidence has supported the role of iron in the development of atherosclerosis. To test whether iron-mediated oxidative stress influences plaque stability, apoliporotein-E (ApoE)-deficient mice (3 months old) were placed on a chow diet or a low-iron diet for 3 months, and the abundance of interstitial collagen and the expression of the matrix degradation-associated enzyme, matrix metalloproteinase-9 (MMP-9), in vascular lesions were assessed. A low-iron diet appeared to reduce iron deposition while substantially increasing collagen content of lesions in mice. Immunostaining demonstrated lower expression of MMP-9 in lesions of iron-restricted animals. Likewise, SDS-PAGE zymography revealed lower gelatinolytic activities in aortic tissues and sera of the same group of animals. When older ApoE-deficient mice (5 months old) received a low-iron diet for 2 months, development of the lesion area was not significantly affected. However, the lesional collagen content was much higher in the iron-restricted group of animals, and MMP-9 expression in aortic tissues from the same group of mice was significantly lower. Treatment of murine J774 macrophages with increasing concentrations of ferric ammonium citrate significantly enhanced the amount of MMP-9 secreted. Together, these data indicate that decreased vascular iron content following dietary iron restriction in ApoE-deficient mice leads to lower matrix degradation capacity and increased plaque stability.     

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.     

Enhanced angiogenesis characteristic of SPARC-null mice disappears with age

The impairment of angiogenesis in aging has been attributed, in part, to alterations in proteins associated with the extracellular matrix (ECM). SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40) is a matricellular protein that regulates endothelial cell function as well as cell-ECM interactions. We have previously shown that angiogenesis, as reflected by fibrovascular invasion into subcutaneously implanted polyvinyl alcohol (PVA) sponges, is increased in SPARC-null mice (6-9 months of age) relative to their wild-type (WT) counterparts. In this study, we define the influence of aging on (a) the expression of SPARC and (b) fibrovascular invasion into sponge implants in SPARC-null and WT mice. The expression of SPARC in fibroblasts and endothelial cells derived from young donors (humans mean age less than 30 years and mice 4-6 months of age) and old donors (humans mean age over 65 years and mice 22-27 months of age) decreased 1.6 to 2.3-fold with age. Analysis of fibrovascular invasion into sponges implanted into old (22-27 months) SPARC-null and WT mice showed no differences in percent area of invasion or collagenous ECM. Moreover, sponges from old SPARC-null and WT mice contained similar levels of VEGF that were significantly lower than those from young (4-6 months) mice. In contrast to fibroblasts from young SPARC-null mice, dermal fibroblasts from old SPARC-null mice did not migrate farther, proliferate faster, or produce greater amounts of VEGF relative to their old WT counterparts. However, when stimulated with TGF-beta1, primary cells isolated from the sponge implants, and dermal fibroblasts from both old SPARC-null and WT mice, showed marked increases in VEGF secretion. These data indicate that aging results in a loss of enhanced angiogenesis in SPARC-null mice, as a result of the detrimental impact of age on cellular functions, collagen deposition, and VEGF synthesis. However, the influence of aging on these processes may be reversed, in part, by growth factor stimulation.