1. Introduction
Nocturnal habits and a cryptic lifestyle have led to the evolution of olfaction as the major communication system in rodents. Mice mark territories and maintain social structure using urine in the form of scent marks (Hurst 1897). Repeated marking in one area often leads to the development of urine pillars. Mouse urine contains large quantities of protein known as MUPs (major urinary protein). MUPs are polymorphic and belong to the lipocalin family of proteins (Flower 1996); they facilitate the gradual release of volatile pheromones into the environment long after the signaller has left the area. They contain a calyx with a -barrel structure that readily binds and transport hydrophobic ligands (Robertson et al. 1998).
Thiazole (2-sec-butyl-4,5-dihydrothiazole) and brevicomin (3,4-dihydro-exo-brevicomin) are two androgen-dependent pheromones (Robertson et al. 1993), which are found only in male mouse urine. They are known to promote inter-male aggression (Novotny et al. 1985) and to stimulate oestrus in female mice (Jemiolo et al. 1985). Disruption of the pheromone-urine complex using chemicals with a higher binding affinity (super-ligands) than the naturally occurring pheromones may provide a subtle means of manipulating problematic populations that rely heavily on urine based communication systems. A previous Defra funded project (VC0411) used QSAR (Quantitative Structure Activity Relationship) to explain the relationship between chemical characteristics of a molecule and its affinity for binding to MUPs. This study showed that increasing the negativity of the ionisation potential (HOMO) improved the binding of ligands to the pheromone-binding site.
Analysis of urine posts by GC-MS revealed quantities of menadione (vitamin K3), believed to have originated from mouse diet. Further analysis using fresh urine, showed that menadione competes with naturally occurring pheromones such as thiazole and brevicomin causing their rapid release from the MUP complex (Robertson et al. 1998). Behavioural observations showed that mice could detect subtle differences between whole urine and urine mixed with menadione (Hurst et al. 1998); their latency to approach displaced scent marks increased in response to the flux of pheromones released. Other studies (Cavaggioni et al. 1990) have shown that some naturally occurring volatiles also have the capacity to preferentially bind to MUP complexes, however their impact on pheromone release has not been quantified.
This study used mice as the model system and aimed to test the potential of the super-ligands identified by the QSAR in silico generated model, to preferentially bind to the MUPs, displacing the naturally bound pheromones thiazole and brevicomin and thereby disrupting the MUP-pheromone communication system.
2. Methods
-
Molecular structure of ligands tested
Menadione
Vitamin K3
Formula C11H8O2
Molecular weight 172.2
Yellow powder
Melting point 105 oC
No detectable odour
(Robertson et al. 1998)
|
|
β Ionone
Formula C13H20O
Molecular weight 192.3
Light yellow liquid
Boiling point 229 0C
Violet odour
(Cavaggioni et al. 1990)
|
|
Geosmin
Formula C12H22O
Molecular weight 182.3
Liquid
Earthy odour
(Cavaggioni et al. 1990)
|
|
Isobutyl methoxypyrazine
Formula C9H14N2O
Molecular weight 166.2
Liquid
Bell pepper odour
|
|
Pseudoephedrine
Formula C19H15NO
Molecular weight 165.2
White crystalline solid
Melting point 118 – 120 oC
No detectable odour
|
|
Ergothioneine
Amino acid
Formula C9H15N3O2S
Molecular weight 229.3
Solid
Melting point 255 – 259 oC
No detectable odour
|
|
Thirty Balb/c male mice were used for urine collection. They were singly housed in RB3 cages and maintained on a 12-hour light : dark cycle. The animals were maintained on a low vitamin K diet and water
ad libitum. Urine was collected by bladder palpation, and stored in eppendorffs at –80
oC until required.
-
Pheromone extraction and GC-MS analysis
Each urine spot (15 µl urine and 15 µl ethanol) was washed with 3 x 90 µl water and the wash collected in an eppendorff. All eppendorffs used had been previously soaked for at least 24 hours in chloroform before use, to minimise leaching of plasticizers into the chloroform-extracted urine. Chloroform (100 µl) was added to the wash and the mixture vortexed for 10 min then left to stand for 1 hour. The samples were spun for 3 min at 1300 rpm and 70 µl of the chloroform layer removed and transferred to a glass sample vial. The sample was further diluted with 80 µl chloroform and 10 µl of the internal standard (hexachlorobenzene; 10 µg/ml)
as an internal standard, to give a final sample volume of 160 µl. Vials were sealed with an airtight cap and stored at –20
oC.
GC-MS analysis was used to measure the concentration of bound thiazole and brevicomin within the artificial scent marks. The instrument used was a Thermoquest GCQ – Thermoquest GC fitted with an ion trap detector, the column was run with a stream of helium at 40 cm/sec. Samples (1 µl) were injected into the column at an oven temperature of 50 oC and held for 1 min. The temperature was ramped at 10 oC/min to 160 oC followed by 25 oC/min to 280 oC and held for 3.2 min. The mass detector was run in full scan mode, with positive ion monitoring of 50 to 290 mass units and electron impact ionisation of 70 eV. As neither brevicomin nor thiazole was commercially available, the concentrations of bound pheromones were quantified by calculating their relative peak area in relation to a known internal reference standard (HCB). Mass spectra of super-ligands were compared to those obtained from analysis of authentic samples.
-
Pheromone release profiles over a 24-hour period
Initial trials were conducted to determine the release profiles of thiazole and brevicomin over a 24-hour period. Artificial scent marks were made in the depressions of a porcelain dimple plates using 15 µl of urine mixed with 15 µl of ethanol and left to air dry for 0 (samples extracted after 3 min), 0.5, 1, 2, 4, 6, 8, 16, 20 and 24 hours, there were six replicates for each time point (Figures 1a & b). All samples were prepared for GC-MS analysis following the same procedure described above. The majority of pheromones were released within the first 10 hours; therefore all future analysis was conducted over a maximum of 6 hours.
Figure 1a: The 24-hour release profile for thiazole, calculated by reference to the peak area of an internal standard HCB.
-
Figure 1b: The 24-hour release profile for brevicomin, calculated by reference to the peak area of an internal standard HCB.
-
-
Pheromone release in the presence and absence of super-ligands
Artificial scent marks were made in the depressions of a porcelain dimple plate using 15 µl of urine mixed with 15 µl super ligand (2.9 mM in ethanol). Control samples were run alongside; here urine was mixed with ethanol only. Samples were left under ambient conditions for 0 hours (samples extracted after 3 min) 0.5, 1, 2 and 6 hours; there were six replicates for each time point before extraction and analysis by GC-MS. Comparisons were made between treatment and controls at each time point using an independent t-test including a test for unequal variances. Analysis of covariance was used to compare the most effective super-ligands; pheromone levels at 0 hours in control samples were used as a covariate to compensate for the variation observed between artificial scent marks.
-
No-choice test to assess the impact of pheromone release on mouse behaviour
Twelve singly housed male Balb/c mice were used throughout the course of the experiment and were given a no-choice test over 4 days to determine their response to pheromone super-ligand treated urine. Two aliquots (30 µl) of the test substance were placed in the central two wells of a porcelain dimple plate; this was covered in fine mesh to prevent mice from coming into direct contact with the artificial scent mark. Plates were left to air dry for 30 min, before being stuck to the wall of the test arena using Velcro strips. Feed hoppers and water bottles were removed from their home cage for the duration of the trial. Combinations of treatment were:
-
Blank (2 x 30 µl of ethanol)
-
Urine (2x (15 µl urine + 15 µl ethanol))
-
Urine & ligand (2 x (15 µl of urine + 15 µl of test ligand))
-
Ligand (2 x (15 µl ethanol and 15 µl of test ligand)
T
he order of presentation was randomly allocated and each animal was given one plate per day. Each mouse was marked with purple dye to allow rapid identification by video surveillance software and equipment. Whilst the test arena
was built within the home cage, mice were held within their nest-box. The trial commenced when the mouse was released from the nest box. Each animal was videoed for 5 minutes after first entering the test arena. Food and water were returned to the home cage once the dimple plate was removed. Video footage was analysed using Ethovision™ software and the test subjects’ latency to approach the test arena and the scent mark area, the number of visits to each zone, and the time spent in each zone quantified. Comparisons between treatments were made using a one-way ANOVA.
Figure 2: Showing the design of the arena for a no-choice test
3. Results
-
Pheromone release in the presence and absence of ligands
Results for thiazole and brevicomin levels for each super-ligand are shown in tables 1 to 4. Thiazole levels were significantly lower in menadione treated urine compared with untreated in all time points after 30 minutes (0.5 hours: t
(10) = 4.97, P = 0.001; 1 hour: t
(10) = 4.23, P = 0.002; 2 hours: t
(5.77) = 6.06, P = 0.001; 6 hours: t
(5.01) = 5.70, P = 0.002). Brevicomin levels were significantly lower in menadione treated urine compared with controls after 30 minutes (0.5 hours: t
(10) = 7.15, P = 0.000; 1 hour: t
(5.56) = 5.95, P = 0.001; 2 hours: t
(5.12) = 9.59, P = 0.000; 6 hours: t
(5) = 5.43, P = 0.003). β ionone (Figure 4a & b) also significantly reduced thiazole levels in treated urine after 30 minutes (0.5 hours: t
(7.19) = 4.65, P = 0.002; 1 hour: t
(10) = 5.05, P = 0.000; 2 hour: t
(10) = 9.32, P = 0.000; 6 hour: t =
(10) = 7.01, P = 0.000). β ionone significantly reduced brevicomin levels after 30 minutes (0.5 hour: t
(5.31) = 8.70, P = 0.000; 1 hour: t
(5.84) = 5.11, P = 0.002; 2 hour: t
(9.24) = 6.71, P = 0.000; 6 hour: t
(10) = 2.59, P = 0.027). All other super-ligands tested were not consistently effective over all time points.
Comparisons were made between menadione and β ionone using analysis of covariance to determine which super-ligand was most effective at displacing naturally occurring pheromones. Menadione displaced significantly more thiazole than β ionone over time (1 hour: F (1, 10) = 11.96, P = 0.009; 2 hour: F (1, 10) = 9.33, P = 0.016; 6 hour: F (1, 10) = 26.80, P = 0.001). Menadione displaced significantly more brevicomin after 0.5 and 1 hours than β ionone (F (1, 10) = 6.95, P = 0.03, F (1, 10) = 24.54, P = 0.01) respectively, after which any differences between super-ligands was not significant. There was no significant difference over time between pheromone release from control urine run along side menadione and β ionone treated urine.
Table 1: Mean relative peak area and standard error for thiazole in the presence and absence of super-ligand. Control and treated were compared at each time point using independent t-tests, significance levels are marked.
Time (Hours)
|
Menadione
|
β ionone
|
Geosmin
|
Control
| Treated |
Control
|
Treated
|
Control
|
Treated
|
0
|
147.31
± 17.51
|
88.99
± 23.06
|
65.84
± 11.01
|
69.23
± 6.35
|
119.70
± 9.09
|
113.94
± 7.45
|
0.5
|
79.14
± 10.55
|
21.65 ±4.78 **
|
45.48
± 3.47
|
27.58
± 1.67 *
|
88.81
± 5.57
|
73.45
± 6.01
|
1
|
77.37
± 14.56
|
15.46
± 1.51 *
|
38.42
± 3.53
|
20.30
± 0.61 **
|
80.32
± 3.66
|
79.81
± 6.13
|
2
|
64.22
± 8.64
|
9.84
± 2.40 **
|
37.67
± 1.64
|
16.59
± 1.56 **
|
78.11
± 2.97
|
68.54
± 2.23 *
|
6
|
49.42
± 8.27
|
2.25
± 0.31 *
|
19.85
± 1.11
|
9.60
± 0.96 **
|
34.99
± 2.33
|
43.34
± 2.17 *
|
* = Significant at the 5 % level
** = Significant at the 1 % level
Table 2:
Mean relative peak area and standard error for thiazole in the presence and absence of super-ligand. Control and treated were compared at each time point using an independent t-tests, significance levels are marked.
Time (Hours)
|
Isobutyl methoxypyrazine
|
Pseudoephedrine
|
Ergothioneine
|
Control
|
Treated
|
Control
|
Treated
|
Control
|
Treated
|
0
|
212.51
± 10.45
|
212.01
± 12.24
|
49.90
± 3.84
|
48.11
± 5.49
|
105.83
± 2.76
|
111.97
± 8.13
|
0.5
|
143.23
± 6.38
|
146.23
± 5.28
|
28.72
± 2.30
|
30.38
± 3.38
|
70.46
± 2.21
|
63.36
± 3.22
|
1
|
151.56
± 6.19
|
135.99
± 9.71
|
26.56
± 1.02
|
28.51
± 1.19
|
81.77
± 3.42
|
63.22
± 2.08 **
|
2
|
104.51
± 2.94
|
116.33
± 4.58
|
26.40
± 1.59
|
23.09
± 0.86
|
57.38
± 1.40
|
64.98
± 3.29
|
6
|
80.20
± 6.94
|
72.04
± 7.74
|
25.90
± 0.56
|
22.04
± 1.53 *
|
53.79
± 2.42
|
56.44
± 2.48
|
* = Significant at the 5 % level
** = Significant at the 1 % level
Table 3: Mean relative peak area and standard error for brevicomin in the presence and absence of super-ligand. Control and treated were compared at each time point using independent t-tests, significance levels are marked.
Time (Hours)
|
Menadione
|
Β ionone
|
Geosmin
|
Control
|
Treated
|
Control
|
Treated
|
Control
|
Treated
|
0
|
67.47
± 6.10
|
51.77
± 12.15
|
18.72
± 2.62
|
20.06
± 1.84
|
27.41
± 2.21
|
30.25
± 1.59
|
0.5
|
12.30
± 1.43
|
1.81
± 0.30 **
|
4.29
± 0.34
|
1.32
± 0.06 **
|
7.78
± 0.69
|
5.60
± 0.47 *
|
1
|
8.40
± 1.17
|
1.28
± 0.28 **
|
2.98
± 0.33
|
1.25
± 0.09 **
|
6.61
±0.91
|
6.00
± 0.43
|
2
|
6.42
± 0.58
|
0.83
± 0.08 **
|
2.94
± 0.18
|
1.44
± 0.13 **
|
7.90
± 0.39
|
6.63
± 0.47
|
6
|
3.30
± 0.61
|
0
± 0 *
|
1.83
± 0.14
|
1.30
± 0.14 *
|
4.52
± 0.31
|
4.45
±0.23
|
* = Significant at the 5 % level
** = Significant at the 1 % level
Table 4: Mean relative peak area and standard error for brevicomin in the presence and absence of super-ligand. Control and treated were compared at each time point using independent t-tests, significance levels are marked.
Time (Hours)
|
Isobutyl methoxypyrazine
|
Pseudoephedrine
|
Ergothioneine
|
Control |
Treated
|
Control
|
Treated
|
Control
|
Treated
|
0
|
77.18
± 4.12
|
70.03
± 4.33
|
18.05
± 1.48
|
17.52
± 2.13
|
22.10
± 0.48
|
24.02
± 1.67
|
0.5
|
19.64
± 0.65
|
14.42
± 1.55 *
|
3.28
± 0.26
|
3.56
± 0.30
|
5.72
± 0.23
|
5.50
± 0.21
|
1
|
22.70
± 1.66
|
16.85
± 1.61 *
|
3.41
± 0.20
|
3.60
± 0.19
|
7.12
± 0.29
|
5.69
± 0.23 *
|
2
|
14.67
± 0.63
|
14.02
± 0.78
|
4.02
± 0.27
|
3.12
± 0.13 *
|
6.10
± 0.25
|
6.67
± 0.29
|
6
|
10.42
± 0.73
|
7.15
± 0.52 *
|
4.04
± 0.17
|
3.21
± 0.29 *
|
5.69
± 0.31
|
6.11
± 0.23
|
* = Significant at the 5 % level, ** = Significant at the 1 % level
Figure 3a: Relative peak area of thiazole at time points 0, 0.5, 1, 2 & 6 hours in the presence and absence (control) of menadione.
Figure 3b: Relative peak area of brevicomin at time points 0, 0.5, 1, 2 & 6 hours in the presence and absence (control) of menadione.
Figure 4a: Relative peak area of thiazole at time points 0, 0.5, 1, 2 & 6 hours in the presence and absence (control) of β ionone.
Figure 4b: Relative peak area of brevicomin at time points 0, 0.5, 1, 2 & 6 hours in the presence and absence (control) of β ionone.
Figure 5a: Relative peak area of thiazole at time points 0, 0.5, 1, 2 & 6 hours in the presence and absence (control) of pseudoephedrine.
Figure 5b: Relative peak area of brevicomin at time points 0, 0.5, 1, 2 & 6 hours in the presence and absence (control) of pseudoephedrine.
No-choice trials using menadione as the test ligand revealed no significant differences in the latency to approach the test arena or the area directly around the scent mark between treatments, neither were there significant differences in the frequency or duration of visits to either zone (Tables 5).
Table 5: Mean and standard error for latency to approach, frequency and duration of visits in the scent mark area in response to a menadione no choice test.
Treatment
|
Latency to
approach (sec)
|
Frequency of visit
|
Duration of visit (sec)
|
Mean
|
SE
|
Mean
|
SE
|
Mean
|
SE
|
Blank
|
9.75
|
4.22
|
15.68
|
2.66
|
18.13
|
3.70
|
Urine
|
16.73
|
6.31
|
16.25
|
2.75
|
24.15
|
4.27
|
Urine + menadione
|
8.82
|
1.79
|
15.42
|
1.57
|
21.38
|
2.75
|
Menadione
|
32.65
|
23.09
|
13.50
|
2.61
|
12.37
|
2.20
|
-
Methods of super-ligand incorporation
During routine analysis of urine extracts, it was discovered that control urine samples had become contaminated by the volatile β ionone; excess β ionone once liberated from the treated scent mark was absorbed by the control (untreated) scent mark. Peak absorption was seen after 1-hour (Figure 6). An
in situ applications strategy (e.g. slow release formulation) strategy was therefore selected as the optimal route of application for this molecule.
Figure 6: Cross contamination of the control (untreated scent mark) by β ionone from treated scent mark. Treated and control scent marks were 10cm apart. Relative peak area determined by reference to a β ionone internal standard at 5 µg / ml.
4. Discussion
Kinetic studies of both the control and super-ligand treated scent marks revealed that all molecules identified found from the literature (e.g. menadione) or predicted by the QSAR model were effective in displacing the MUPs pheromonal cargo. As expected menadione, our positive control, was effective in displacing both thiazole and brevicomin, as was β ionone, but to a lesser degree. There was some evidence of displacement initiated by geosmin, isobutyl-methoxypyrazine, pseudoephedrine and ergothioneine, but this was not consistent over all the time points.
Behavioural responses to the differences in pheromone release in the presence and absence of menadione have been detected in a choice test (Hurst et al. 1998). However, because of the volatility of many of our super-ligands, and hence the risk of contaminating the control, we considered a choice test to be inappropriate. Behavioural changes in response to pheromone displacement by our positive control, menadione, could not be detected in our no-choice test and therefore we did not test any of predicted ligands using this test method. During behavioural observations, there was no evidence that this method of disrupting the social communication between mice caused undue stress to the individuals involved. Further research is required to develop a behavioural choice assay which avoids cross contamination between test and control scent marks.
The ability of β ionone to displace pheromones and our discovery that it readily contaminates untreated scent marks over a relatively wide area makes this a candidate material for disrupting scent mark communication although its practical utility remains to be proven.
The model developed in project VC0411 requires further refinement to increase its effectiveness in predicting putative super-ligands. New and more powerful programmes are constantly under development and CSL has very recently acquired DRAGON which calculates a large number of varied molecular descriptors that can help in the design of more potent ligands.
5. References
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Flower, D. R. (1996) The lipocalin protein family: Structure and function. Biochemical Journal, 318, 1-14.
Hurst, J., Robertson, D. H. L. & Tollady, U. (1998) Proteins in urine scent marks of male house mice extend the longevity of olfactory signals. Animal Behaviour, 55, 1289-1297.
Hurst, J. L. (1897) The functions of urine marking in a free-living population of house mice,
Mus domesticus Rutty.
Animal Behaviour,
35, 1433-1442.
Jemiolo, B., Alberts, J., Sochinski-Wiggins, S., Harvey, S. & Novotny, M. (1985) Behavioural and endocrine responses of female mice to synthetic analogs of volatile compounds in male urine.
Animal Behaviour,
33, 1114-1118.
Novotny, M., Harvey, S., Jemiolo, B. & Alberts, J. (1985) Synthetic pheromones that promote inter-male aggression in mice.
Proc.Natl Acad.Sci.USA,
82, 2059-2061.
Robertson, D., Hurst, J., Hubbard, S., Gaskell, S. J. & Beynon, R. (1998) Ligands of urinary lipocalins from the mouse: Uptake of environmentally derived chemicals.
Journal of Chemical Ecology,
24, 1127-1140.
Robertson, D. H. L., Beynon, R. J. & Evershed, R. P. (1993) Extraction, characterization, and binding analysis of 2 pheromonally active ligands associated with major urinary protein of house mouse (
Mus musculus).
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19, 1405-1416.