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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials And Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References

Objectives

To investigate the association between dog hair nicotine concentration and owner-reported exposure to environmental tobacco smoke to establish whether dogs are exposed to significant, detectable amounts of environmental tobacco smoke in the home.

Methods

Hair was collected from 23 dogs exposed to environmental tobacco smoke and from 15 dogs reportedly not exposed to environmental tobacco smoke. Hair was washed to remove adhered nicotine, digested in 1 M NaOH and extracted using solid phase extraction. Nicotine concentration was measured by high-resolution mass spectrometry. Results were analysed using a Kruskall-Wallis test and post hoc pair-wise comparisons using a Mann–Whitney test to assess significance between exposure groups.

Results

The different exposure groups were significantly different (P < 0·001) for both hair and surface nicotine. Pair-wise comparisons were significant at P < 0·05 for all categories except unexposed and occasionally exposed groups (P = 0·076).

Clinical Significance

Dog hair nicotine concentration appears to be strongly associated with reported exposure to environmental tobacco smoke. The range and median of hair nicotine concentration in dogs exposed to environmental tobacco smoke was similar to those reported in children. This suggests that dog hair could provide a useful method of determining the amount of environmental tobacco smoke exposure in all environments common to pets and children.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials And Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References

Exposure to environmental tobacco smoke (ETS) through second-hand smoke (SHS) can be considerable in both adults and children (Pichini and others 1997, Kim and others 2008, Wipfli and others 2008) and is a known cause of smoking-related disease. In pets, the association between ETS and disease has been difficult to prove as assessed by questionnaire-based studies (Pichini and others 1997, Bukowski and others 1998, Reif and others 1998, Bertone and others 2002, 2003). However, in dogs, ETS exposure appears to increase the relative risk of cancer of the lung (Reif and others 1992), nasal cavity and paranasal sinuses (Reif and others 1998). An association with chronic coughing was not confirmed (Hawkins and others 2010), although exposure to ETS was associated with increased populations of macrophages and lymphocytes and macrophage anthracosis in bronchioalvealor lavage fluid collected from Yorkshire terriers exposed to ETS (Roza and Viegas 2007).

Previous studies have shown that dogs excrete the nicotine by-product cotinine in the urine (Bertone-Johnson and others 2008). This method of assessing SHS exposure only provides evidence of recent exposure and requires a urine sample which can be difficult to obtain by free-catch methods in some patients. In humans, hair nicotine concentrations (HNC) is a useful biomarker of ETS exposure (Eliopoulos and others 1994, -Pichini and others 1997, Al-Delaimy and others 2000, 2002, Kim and others 2008, Wipfli and others 2008) that occurred over the previous 2 to 3 months (Uematsu 1993). Dogs have a similar rate of hair growth to humans (extrapolated from Muller and others 1989) and the HNC should therefore reflect a similar time frame of ETS exposure, although in non-shedding breeds hair-growth rate may be altered. As environmental nicotine adheres to the surface of hair, the methods for assessing HNC in humans involves washing the hair before analysis. This surface nicotine may be important in dogs as self-grooming will allow at least some of this surface nicotine to be internalised. Exposure to ETS may therefore result in greater HNC in pets than that seen in humans exposed to ETS. The closeness of interactions between children and smokers (Wipfli and others 2008) are thought to explain the 35% higher HNC (Kim and others 2008) seen in exposed children, and this effect is likely to be mirrored by many pet-owner relationships.

The aims of this study were first to establish whether dogs are exposed to significant, detectable amounts of ETS in the home and, secondly, to investigate the association between dog HNC and owner-reported exposure to ETS.

Materials And Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials And Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References

Hair samples

Hair was collected from 38 dogs with the owners’ consent. Dogs were presented to the University of Glasgow's Small Animal Hospital or the PDSA PetAid Hospital, Shamrock Street, Glasgow. Additional samples were collected from dogs owned by staff and students of the University of Glasgow. Samples were collected from any dogs for which the owners gave consent but were latterly selected based on the owner-reported exposure to ensure that the study recruited approximately even numbers of dogs never exposed to ETS and regularly exposed to ETS. Since the speed of hair growth is affected by anatomical site (Diaz and others 2004), all samples were collected from the neck region. The neck collection site was selected as a common site for clipping before blood sample collection, thereby allowing results from this study to be compared to those obtained in larger-scale studies in the future. Although recent studies have suggested that photoperiod has little effect on hair-growth rates in dogs kept indoors (Diaz and others 2004), all samples were collected within a three-month period to minimise any effects that photoperiod might have on the rate of hair growth and therefore nicotine incorporation (Diaz and others 2004). The hair was stored in sealed envelopes and kept in a smoke-free environment to ensure that no further ETS exposure, which could affect surface nicotine concentrations, occurred after hair collection. The signalment of each dog was recorded.

Questionnaire estimating ETS exposure

For each of the samples collected, owners completed a brief questionnaire detailing the amount of ETS they believed their dog had been exposed to in the previous three months. The questionnaire was modelled on similar studies in humans (Al-Delaimy and others 2002). The questionnaire included details of the -location of exposure: during close contact with owner, in the dog's home, during car travel, in friend's or family's home or outdoors. In each case, the owners graded the exposure as regular, occasional or never. A copy of the questionnaire can be obtained from the corresponding author on request. Dogs were then grouped into those never exposed (group 0), those exposed occasionally (group 1) and those exposed regularly (group 2). Occasional exposure was defined as occasional, and not regular, exposure in the dog's home, during car travel and in friend's or family's home, but included dogs exposed regularly or occasionally when outdoors. Regular exposure was defined as reported regular exposure in the dog's home. Some of the dogs reported to have regular exposure in the house also had regular or occasional exposure during car travel and in friend's or family's homes.

Measurement of HNCs

Samples of dog hair (ca 30 mg) were washed with 2 mL of methanol by sonicating for 15 minutes at room temperature. The methanol was removed and the washings were retained for analysis. The hair was then treated with 1 M NaOH (1 mL containing 1 µg of 2 H4-nicotine) at 50 °C for 24 hours. The samples were then loaded onto Strata X columns (30 mg, Phenomenex UK) which had been prewashed with 1 mL of NaOH. The columns were then washed with 2 mL of water and then eluted with 1 mL of acetonitrile/water (95:5) containing 3·25 mM ammonium acetate. The extracts were analysed by using an Exactive Mass Spectrometer (ThermoElectron UK) operated in positive-ion ESI mode with a needle voltage of 4·5 kV, a heated capillary temperature of 275 °C, sheath gas flow of 50 arbitrary units and auxiliary gas flow of 17 arbitrary units. Chromatography was carried out using a Dionex 3000 binary HPLC pump fitted with a ZICHILIC column (150 × 4·6 mm 5 µm particle size, Hichrom UK), a gradient was used with a flow rate of 0·5 mL/min. Mobile phase A was 0·1% formic acid in water and mobile phase B was 0·1% formic acid in acetonitrile. The gradient was as follows: 60% B 0 min to 20% B at 20 min followed by re-equilibration for 10 minutes. The method was calibrated in the range 0·05, 0·1, 0·2, 0·4, 0·8, 1·6 ng/mL, and the lowest level of detection was around 1 ng/mL. This method and the validation are described in more detail by Bawazeer and others (2012). The precision of the method was tested by repeat analysis. Nicotine was not detected in blank samples of hair. Nicotine concentrations integrated within the hair (HNC) as well as that extracted by the washing process were measured.

Statistical analysis

Summary statistical analysis was calculated for HNC and for wash nicotine for dogs grouped into three categories of owner-reported SHS exposure. Given the evident skewed nature of the data (see boxplots in Fig 1), a non-parametric statistical approach (the Kruskall-Wallis test) was used to test the null hypothesis of no difference in HNC between owner-reported exposure groups. Post hoc pair-wise comparisons using a Mann–Whitney test were conducted to assess the significance of differences between individual exposure groups. All analyses were conducted in Minitab v 16. Statistical significance was considered to be P < 0·05.

image

Figure 1. Hair nicotine and wash nicotine concentrations for dogs with no exposure to environmental tobacco smoke (ETS) (0), occasional exposure to ETS (1) and regular exposure to ETS (2). The boxes represent the central 50% of the data, i.e. from the 25th to the 75th percentile, with the horizontal line within the box denoting the median; * denotes outliers

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials And Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References

Thirty-eight dogs were included in the study. The median age was 7 years (ranging from 4 months to 15 years). Eighteen dogs were male and 20 were female. There were 19 breeds represented including 10 crossbreeds, 5 Labrador retrievers, 3 Staffordshire bull terriers and the remainder a wide variety of breeds. Three dogs were considered to have a non-shedding coat [one standard poodle and two poodle crossbreds (jackadoodle and labradoodle)]. All non-shedding breed dogs were in group 0.

Twenty-three dogs were reportedly exposed to ETS, of which 7 dogs had reported occasional ETS exposure (group 1) and 16 dogs had regular ETS exposure (group 2). Fifteen dogs had no reported ETS exposure (group 0). In the 16 dogs reportedly regularly exposed to ETS, HNC ranged from 0·1093 to 11·306 ng/mg (median 0·57 ng/mg), while the wash nicotine concentration ranged from 0·0 to 9·8026 ng/mg (median 0·91 ng/mg). In the seven dogs occasionally exposed to ETS, HNC ranged from 0 to 0·1858 ng/mg (median 0·09 ng/mg) and wash nicotine concentration ranged from 0·0046 to 0·8124 ng/mg (median 0·08 ng/mg). In the 15 dogs not exposed to ETS, HNC ranged from 0 to 3·083 ng/mg (median 0·01 ng/mg) and wash nicotine concentration ranged from 0 to 0·155 ng/mg (median 0·01 ng/mg). Boxplots of the distribution of hair and wash nicotine concentrations in the three exposure categories are shown in Fig 1. There was a statistically significant difference (P < 0·001) in both hair and wash nicotine between all groups, suggesting the potential utility of these methods for monitoring ETS exposure. All pair-wise comparisons between individual exposure categories were significant at P < 0·05 except that between the unexposed and occasionally exposed groups for HNC (P = 0·076).

Analysis of the owner questionnaires collected at the time of hair sampling, (Table 1) revealed that 87% of dogs were reportedly exposed to ETS in their own homes and 74% of dogs were reportedly exposed during close contact with a smoker. In -addition, 74% of owners reported that their dogs were reportedly exposed to ETS outdoors.

Table 1. Potential locations where dogs in this study are exposed to ETS as reported by their owners
Reported locations of ETS exposurePercentage of dogs exposed (n = 23)
  1. ETS Environmental tobacco smoke

Close contact with owner74
In pet's home87
During car travel35
In family or friends’ home39
Outdoors74

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials And Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References

This study shows that dog HNC appears to be closely associated with reported exposure to ETS. Both the HNC and the surface (wash) nicotine were significantly different between all groups, except the unexposed and occasionally exposed dogs when assessed by pair-wise comparisons. Occasional ETS exposure was associated with reduced median nicotine concentrations in both hair and wash compared to dogs with reported regular ETS exposure. This suggests that reducing exposure by smoking outdoors or by reducing the amount of direct contact with ETS can significantly reduce the amount of HNC in dogs. However, the HNC of dogs exposed occasionally to ETS remains greater than that of dogs that are never exposed.

Hair nicotine was found in 11 of 15 dogs reportedly not exposed to ETS, which is similar to the studies reported in humans (Pichini and others 1997, Al-Delaimy and others 2002). It is possible that exposure is taking place through alternative routes not recognised by the owners, such as environmental pollution. One dog with no reported ETS exposure had high HNC (3·0834 ng/mg) which would be more consistent with regular exposure. This dog was only 16 weeks old at the time of sampling and was a standard poodle. Given the age of the puppy it may have had exposure to ETS in its neonatal environment, as puppies are usually re-homed at 8 to 12 weeks of age and the HNC could therefore reflect nicotine exposure in the first half of the puppy's life. Unfortunately, although the questionnaire asked owners to record exposure over the previous 3 months, it did not specifically request information on the ownership of the dog over that timeframe.

Another potential explanation for the result is in the dog's hair type. Poodles are known for their non-shedding hair coat which might increase long-term nicotine build-up in older poodles. However, the age of this dog meant that unless nicotine build-up started during gestation the timeframe for potential exposure is limited by its age. The two other dogs that were poodle crossbreds (jackadoodle and labradoodle) were both also in group 0 but had low HNC (0·0225 ng/mg and 0·0049 ng/mg, respectively), suggesting that non-shedding hair type alone is unlikely to be responsible. The inclusion of these non-shedding breeds in group 0 may have limited the detection of differences between the dogs never exposed and the other two groups. The poodle's hair colour was not recorded; however, in rats, it has been suggested that nicotine has a higher affinity to melanin, with increased HNC occurring in pigmented hair compared to albino hair (−Gerstenberg and others 1995). The dogs sampled in this study did have a wide range of coat colours. However, since we did not actively sample hair from different coat colour regions, the data collected cannot establish if this difference is also present in dogs. Future studies should ensure that coat colour is recorded and establish whether coat colour variations affect HNC by the collection of samples of differing coat colours from the same dog.

All hair samples were collected from the neck region to ensure consistency of sampling site as hair growth varies with anatomical location (Diaz and others 2004). Similarly, the speed of hair growth in dogs varies according to final hair length, with longer hair growing faster (Diaz and others 2004). Although breed was recorded, this preliminary study is too small to determine whether breed or hair type affects the amount of nicotine incorporated into the hair. The wash nicotine and HNC showed good correlation on rank analysis, although differences in the amount of adhered nicotine could be associated with removal by washing, swimming or grooming before sampling. Information about washing and swimming frequency would be useful to determine what effect this coat cleaning has on the HNC and therefore whether ingestion during self-grooming increases HNC.

The median HNC identified in dogs regularly exposed to ETS (0·57 ng/mg) was similar to those reported in children (0·68 ng/mg) and women (0·4 ng/mg) exposed to SHS (Wipfli and others 2008). The range of HNC in dogs exposed to ETS is wider than that reported in Finnish women (0·9 to 2 ng/mg) exposed to SHS (Jaakkola and others 2001). In the study by Wipfli and others (2008), children under five years had median HNC twice that of children over five years. This probably reflects the close contact between young children and smokers in the home. The data published by Wipfli and others (2008) suggest that HNCs up to 10 ng/mg were recorded in these children. These figures are similar to those obtained in this study. Human studies have also shown that nicotine is present in the hair of children not exposed to ETS (Pichini and others 1997). These findings together suggest that dogs experience similar ETS exposure in the home as do children. The lack of data on hair coat type and colour, frequency of washing/swimming and environment limit the conclusions that can be drawn from this pilot study, although we can confirm that HNC in dogs appears to mirror that recorded in children. By establishing the associations that these variables have with dog HNC, we could establish whether dog hair would provide a useful method of determining the amount of ETS exposure in all environments common to pets and children.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials And Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References

We thank Scottish Life Sciences Alliance for funding the Exactive mass spectrometer, British Small Animal Veterinary Association's Petsavers Charity for funding the sample analysis and the PDSA for allowing samples to be collected from the dogs presented to their PDSA PetAid Hospital, Glasgow (Shamrock Street).

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials And Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References

The authors confirm that all individuals personally acknowledged have given their permission to be listed. The authors confirm that all co-authors have given their permission to be listed.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials And Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest
  9. References
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