Assessment of Heavy Metal and Mineral Levels in

Hair Samples from Dogs with Mammary Neoplasms

Emanuela BADEA1*, Gheorghe Valentin GORAN1, Cristina ȚOCA2, Victor CRIVINEANU1

1
Faculty of Veterinary Medicine, UASVM of Bucharest, 105 Splaiul Independenței, 050097, 5th district,
Romania, EU
2
IDAH of Bucharest, 63 Doctor Staicovici, 050557, 5th district, Romania, EU
* corresponding author: emanuela.badea@gmail.com

Bulletin UASVM Food Science and Technology 75(1)/2018

ISSN-L 2344-2344; Print ISSN 2344-2344; Electronic ISSN 2344-5300
DOI: 10.15835/buasvmcn-fst: 0007

ABSTRACT
Neoplasms involve abnormal tissue growths developing in an uncoordinated, persistent manner, faster than
adjacent normal tissues. Several researchers have studied the possible implications of heavy metals and mineral
levels on human mammary neoplasms using hair analysis. The study’s objective was to assess the levels of heavy
metals and other mineral elements in dogs suffering from mammary neoplasms. Hair samples were analyzed
by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). All analyzed elements registered higher levels in
clinically healthy dogs. The interaction between health status and keeping conditions significantly influenced the
levels of Al, Ca, Fe, Mg, Ni, Co, Pb, and V. Concurrently, dogs with mammary neoplasms living outdoors registered
the highest levels for most minerals, compared to dogs with mammary neoplasms living indoors, suggesting a
possible implication of pollution.

Keywords: heavy metals, minerals, mammary neoplasms, dogs, hair.

INTRODUCTION researchers have studied the possible implications

Neoplasms are a disease of the genome, involv-
ing a pathological activity concerning proliferation,
differentiation and death of particular cells, abnor-
mal tissue growths developing in an uncoordinated,
persistent manner, faster than adjacent normal tis-
sues. Neoplasms can be either benign or malignant,
with different causes determining the onset of dif-
ferent types of cancer. (Dobson, and Lascelles ,2011;
Meuten, 2017; Morris, and Dobson, 2001) Mamma-
ry gland tumors are the most commonly diagnosed
neoplasms in female dogs (Pérez et al., 1998; Phi-
libert et al., 2003; Sarli et al., 2002; Yamagami et al.,
1996), some researchers stating that they account
for 70% of all cancer cases. (Merlo et al., 2008). In-
tact status is the most common cause of mammary
cancer (Dorn et al., 1968; Schneider et al., 1969;
Sorenmo et al., 2000), along with increased age and
progestagen treatment (Perez et al., 2000). Several
of heavy metals and mineral levels on human mam-
mary neoplasms by hair analysis. (Joo et al., 2009;
Pasha et al., 2010; Wang et al., 2006; Yasuda et al.,
2009)
The study’s objective was to assess the levels
of heavy metals and other mineral elements in
dogs suffering from mammary neoplasms.
MATERIALS AND METHODS
The study was conducted on 30 female dogs,
divided into two groups: the study group (SG)
consisting of dogs suffering from mammary
neoplasms (n = 15), and the control group (CG)
consisting of clinically healthy dogs (n = 15). All
dogs suffering from mammary neoplasms were
post-op diagnosed with adenocarcinoma, using
histopathological examination. In the SG, all dogs
were above 7 years of age, of which 7 dogs lived
 BADEA et al.

indoors, and 8 lived outdoors. All dogs in the CG
were above 5 years of age, of which 5 dogs lived
indoors, and 10 lived outdoors. Based on keeping
conditions, the groups were further divided into
SGI (study group dogs living indoors), SGO (study
group dogs living outdoors), and CGI (control
group dogs living indoors), CGO (control group
dogs living outdoors), respectively.
All hair samples were collected from the flank
region, placed in disposable paper envelopes,
labeled, and transported to the laboratory. The
samples were degreased, washed, and rinsed.
Each sample weighed roughly 0.5g, and was
digested using 5 ml HNO3 65% Suprapur PA and
1 ml HCl fuming 37% Selectipur PA, then diluted
to 10 ml with ultrapure water and analysed by
Inductively Coupled Plasma Mass Spectrometry
(ICP-MS). Samples were analysed using Perkin -
Elmer - Elan DRC II ICP-MS spectrometer (RF1100
W; reading time 30 s, washing time 30 s, nebuliser
gas flow 0.5 L min-1; auxiliary gas flow 0.5 L min-
1; sample injection pump flow 50 rpm). Results for
each sample are the average of three successive
measurements. (Tudoreanu, 2005) Calibration
curves were developed using standard solutions
of 0.001 ppm, 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm,
10 ppm, 50 ppm obtained by dilution from a multi-
element ICP MERCK standard containing 1000 ml
L-1 of heavy metals (Al, As, Cd, Cr, Ni, Pb, Sr) and
other mineral elements (Ca, Co, Cu, Fe, K, Mg, Mn,
Se, V, Zn).
Statistical analysis was performed using
VassarStats: Website for Statistical Computation
(http://vassarstats.net/). One-Way ANOVA was
performed for all samples’ heavy metal and
mineral concentrations.
RESULTS AND DISCUSSION
Mean heavy metal and mineral levels in dogs
suffering from mammary adenocarcinoma and
clinically healthy dogs, along with p-value resulting
from One-Way ANOVA, are presented in Tab. 1.

Table 1. Study group and Control group heavy metal and other mineral elements mean concentrations
(mg•kg-1)

Element Study group Control group p-value*

Al 66.00 215.14 0.29
As BDL** 0.84 -
Ca 1045.81 2300.22 0.18
Cd 0.02 0.03 0.72
Co 0.17 0.30 0.50
Cr 2.66 3.79 <0.05
Cu 11.74 12.30 0.83
Fe 106.16 302.43 0.29
K 631.25 1199.77 0.29
Mg 133.23 288.78 0.13
Mn 4.17 13.32 0.53
Ni 0.76 1.01 0.44
Pb 0.74 1.25 0.28
Se 0.20 0.65 0.60
Sr 1.67 4.49 0.09
V 1.28 2.95 <0.01
Zn 101.74 117.44 0.49

* p-value resulting from One-Way ANOVA

** BDL – below method’s detection limit

Bulletin UASVM Food Science and Technology 75(1) / 2018

Assessment of Heavy Metal and Mineral Levels in Hair Samples from Dogs with Mammary Neoplasms

All analyzed elements were insignificantly
higher in clinically healthy dogs compared to the
study group, with only chromium (p < 0.05) and
vanadium (p < 0.01) being significantly higher.
For comparison with other findings in
scientific literature related to heavy metal and
other mineral elements levels in hair samples
taken from patients with breast cancer, Tab. 2
reveals what methods of element quantification
other researchers used, along with their results.
Al (Romanowicz-Makowska et al., 2011), As
(Benderli Cihan et al., 2011; Zhang et al., 2007),
Ca (Benderli Cihan et al., 2011), Cd (Ionescu et al.,
2006; Benderli Cihan et al., 2011; Romanowicz-
Makowska et al., 2011), Co (Benderli Cihan et al.,
2011), Cr (Ionescu et al., 2006), Cu (Geraki et al.,
2004; Geraki et al., 2002; Margalioth et al., 1983;
Mulay et al., 1971), Fe (Geraki et al., 2004; Geraki
et al., 2002; Ionescu et al., 2006), K (Geraki K et
al., 2004), Mg (Santoliquido et al., 1976; Mulay et
al., 1971), Mn (Mulay et al., 1971; Benderli Cihan
et al., 2011), Ni (Ionescu et al., 2006; Benderli
Cihan et al., 2011; Rizk and Sky-Peck, 1984), Pb
(Ionescu et al., 2006; Benderli Cihan et al., 2011),
Se (Benderli Cihan et al., 2011; Rizk and Sky-Peck,
1984), Sr (Rizk and Sky-Peck, 1984), Zn (Kelleher
et al., 2009; Geraki et al., 2004; Geraki et al., 2002;
Margalioth et al., 1983; Santoliquido et al., 1976;
Mulay et al., 1971; Ionescu et al., 2006; Benderli
Cihan et al., 2011) registered higher levels in
human cancerous breast tissue compared to
noncancerous breast tissue. The present study
found that all these elements were decreased
in hair samples taken from dogs suffering from
mammary cancer, compared to hair samples taken
from clinically healthy dog. These findings suggest
that the analyzed elements could not reach the hair
in order to accumulate, as they might be retained
in the cancerous mammary tissue.
Additionally, Cd (Johnson et al., 2003; Silva
et al., 2012; Byrne et al., 2013) and Cr (Martin
et al., 2003) form a high-affinity complex with
the hormone binding domain of the estrogen
receptors in the mammary gland. These findings

Table 2. Heavy metal and other mineral elements levels in hair samples taken from patients with breast
cancer compared to clinically healthy patients

Higher Higher in
Type of hair The same in
Method in cancer control References
sample both groups
patients samples
Flame atomic
Skrajnowska
Rat absorption Fe, Zn Ca, Mg
et al. (2014)
spectroscopy
Flame atomic Ca, Cd, Co, Cr,
Pasha et al.
Human absorption Cu, K, Mg, Mn, Al, Fe
(2010)
spectroscopy Ni, Pb, Sr, Zn
Ca, Cr, Cu, Fe,
Joo et al.
Human Mass spectrometry Al, As, K Mg, Mn, Pb, Se, Cd
(2009)
Zn
Inductively coupled
Al, Ca, Cd, Co, Cr, K, Mg, Mn, Ni, Yasuda et al.
Human plasma mass
Cu, Fe, Pb, Zn Se, V (2009)
spectrometry
Atomic absorption
spectrometer with Memon et al.
Human Zn
deuterium background (2007)
correction
Flame atomic
Wang et al.
Human absorption Cu, Fe Ca, Mg, Zn
(2006)
spectroscopy
Graphite furnace
Kilic et al.
Human atomic absorption Cr Mn
(2004)
spectroscopy

Bulletin UASVM Food Science and Technology 75(1) / 2018

 BADEA et al.

further suggest that Cd and Cr cannot accumulate
in hair because they are retained in the mammary
tissue.
Ca also has a reduced intestinal absorption and
elevated endogenous fecal concentration in human
patients suffering from breast cancer (Coombes et
al., 1977), providing additional evidence that Ca
cannot accumulate in cancer patient’s hair.
Vanadium has the potential of being an
antitumor agent (Manna et al., 2011; Ray et al.,
2007; Bishayee et al., 2000). This suggests that
dogs with cancer lacked V’s protection, thus
leading it to also not be found in hair, compared

Figure 1. Mean concentrations of Ca and K based on health status and keeping conditions
(SGI = study group living indoors; SGO = study group living outdoors;
CGI = control group living indoors; CGO = control group living outdoors)

Figure 2. Mean concentrations of Al, Fe, Mg, and Zn based on health status and keeping conditions
(SGI = study group living indoors; SGO = study group living outdoors;
CGI = control group living indoors; CGO = control group living outdoors)

Bulletin UASVM Food Science and Technology 75(1) / 2018

Assessment of Heavy Metal and Mineral Levels in Hair Samples from Dogs with Mammary Neoplasms

to the significantly higher levels of V (p < 0.01)
registered in the hair of clinically healthy dogs, as
the present study revealed.
Mean concentrations of heavy metals and
other mineral elements based on health status and
keeping conditions are shown in Fig. 1-4. Between
SGI, SGO, CGI, and CGO, CGI overall registered the
highest levels for most heavy metals and other
mineral elements: Al, As, Ca, Co, Cr, Fe, K, Mg, Mn,
Ni, Pb, Sr, and V. The remaining elements (Cd, Cu,
Se, Zn) were higher in CGO.
Between SGI and SGO, SGI registered the
highest levels of K, all other elements being higher
in the SGO.

Figure 3. Mean concentrations of Cu, Cr, Mn, Ni, Sr, and V based on health status and keeping conditions
(SGI = study group living indoors; SGO = study group living outdoors;
CGI = control group living indoors; CGO = control group living outdoors)

Figure 4. Mean concentrations of As, Cd, Co, Pb, and Se based on health status and keeping conditions
(SGI = study group living indoors; SGO = study group living outdoors;
CGI = control group living indoors; CGO = control group living outdoors)

Bulletin UASVM Food Science and Technology 75(1) / 2018


Other researchers have concluded that heavy
metals and other mineral elements register higher
levels in the hair of individuals who have been in
contact with polluted environments (Barana et al.,
2013; Skibniewski et al., 2013; Tomza-Marciniak
et al., 2012; Skibniewska et al., 2011; Miranda et
al., 2004).
The results obtained in this study partially
confirm other findings from the scientific literature
regarding higher levels of heavy metals and other
mineral elements being found in individuals from
polluted environments.
CONCLUSIONS
All analyzed elements were elevated in
clinically healthy dogs’ hair compared to dogs
with mammary adenocarcinoma, as they might
be retained in the cancerous mammary tissue,
leading them to not accumulate in the hair of dogs
with cancer.
The absence of vanadium’s protecting
effect against mammary cancer may explain the
significantly lower levels found in the hair of dogs
with mammary adenocarcinoma.
Between dogs with cancer living indoors
and those living outdoors, with the exception
of K, all analyzed elements were higher in the
outdoor dogs, suggesting a possible implication of
environmental pollution.
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