Therapeutic Effect of Hydrogen Injected Subcutaneously on Onion Poisoned Dogs

Abstract

The purpose of this study was to investigate the therapeutic effect of hydrogen on the therapy of onion poisoned dogs.
A total of 16 adult beagle dogs were divided into two groups (control and hydrogen) and all were fed dehydrated onion powder at the dose of 10 g/kg for three days. The dogs of the experimental group were given subcutaneous injection of 0.2 mL/kg of hydrogen for 12 days after making the poisoned model successful. Blood samples were collected before feeding onions, one day before injecting hydrogen, and 2 h after the injection of hydrogen on days 1, 3, 5, 7, 9, and 12. Control dogs were not treated with hydrogen.
The levels of leukocyte production, anaemia, red blood cell degeneration which was reflected by the values of Heinz body count, haemolytic ratio, and oxidative products in hydrogen treated group were lower than in control dogs on some days. The capacity of medullary haematopoiesis that was based on reticulocyte counts, and the antioxidation in hydrogen group were higher compared with control group. However, the differences in renal function were not obvious in both groups.
Accordingly, it was concluded that subcutaneous injection of hydrogen could alleviate the symptoms in onion poisoned dogs.

Full text

J Vet Res 61, 527-533, 2017
DOI:10.1515/jvetres-2017-0068
DE GRUYTER
OPEN
DE
G
Therapeutic effect of hydrogen
injected subcutaneously on onion poisoned dogs
Jinghua Zhao1, Ming Zhang1, Yue Li1,
Zhiheng Zhang1, Mingzi Chen1, Tao Liu1,
Jiantao Zhang1, Anshan Shan2
1Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, College of Veterinary Medicine,
2College of Animal Science and Technology, Northeast Agriculture University, Harbin 150030, China
zhangjiantao@neau.edu.cn; shanasvip@163.com
Received: July 11, 2017 Accepted: December 6, 2017
Abstract
Introduction: The purpose of this study was to investigate the therapeutic effect of hydrogen on the therapy of onion
poisoned dogs. Material and Methods: A total of 16 adult beagle dogs were divided into two groups (control and hydrogen) and
all were fed dehydrated onion powder at the dose of 10 g/kg for three days. The dogs of the experimental group were given
subcutaneous injection of 0.2 mL/kg of hydrogen for 12 days after making the poisoned model successful. Blood samples were
collected before feeding onions, one day before injecting hydrogen, and 2 h after the injection of hydrogen on days 1, 3, 5, 7, 9,
and 12. Control dogs were not treated with hydrogen. Results: The levels of leukocyte production, anaemia, red blood cell
degeneration which was reflected by the values of Heinz body count, haemolytic ratio, and oxidative products in hydrogen
treated group were lower than in control dogs on some days. The capacity of medullary haematopoiesis that was based on
reticulocyte counts, and the antioxidation in hydrogen group were higher compared with control group. However, the differences
in renal function were not obvious in both groups. Conclusion: Accordingly, it was concluded that subcutaneous injection of
hydrogen could alleviate the symptoms in onion poisoned dogs.
Keywords: dogs, onion poisoning, hydrogen, therapy.
Introduction
The onions have been widely recognised to possess
some medicinal properties, such as promoting blood
circulation, anti-bacterial and anti-inflammation
properties, aside from their nutritional value and taste
benefits for humans (6, 26). However, some animal
species may show symptoms of poisoning after eating
onions. Among animal species, cats and dogs are
relatively susceptible to onion-induced oxidative
damage, followed by cattle, horses, sheep, goats, rats,
and mice, in order of increasing resistance (8, 21).
The toxic elements in onions are mainly n-propyl
disulfide (5, 22) and sodium n-propyl thiosulfate (18,
25), which can increase the counts of Heinz bodies and
decrease glutathione concentration in erythrocytes. The
mechanism that causes onion poisoning in dogs is
attributed to damage of the antioxidant system in
erythrocytes, causing series of pathological changes in
erythrocyte membrane and intracellular materials, and
finally leading to haemolytic anaemia (24). Clinical
symptoms mainly include red urine, a decrease in red
blood cells, as well as a substantial increase in Heinz
bodies and reticulocyte number. It should be noted that if
the severely poisoned dogs are not treated appropriately,
they will die eventually. It is reported that raw onions,
cooked onions, dehydrated onions, and crushed onions
are all potentially toxic for dogs (7).
Hydrogen is the most abundant element in the
universe. Until recently the knowledge on potential
biological application of hydrogen was scarce. In 2007,
Ohsawa et al. (20) reported that 2% inhalation of
hydrogen gas could ameliorate significantly the cerebral
ischaemia reperfusion injury in rats, and it was based on
a selective decrease in hydroxyl free radical (•OH) and
peroxynitrite (ONOO–), which were much more reactive
than other reactive oxygen species (ROS) (20). The
study caused quickly a widespread concern and scientists
had made great progress in medical application of
hydrogen molecule. Many published papers have
© 2017 J. Zhao et al. This is an open access article distributed under the Creative Commons Attribution-
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528 J. Zhao et al./J Vet Res/61 (2017) 527-533
indicated that hydrogen has numerous biological effects,
such as selective antioxidation (20), anti-infection (10),
anti-tumour (17), and anti-radiation (14), and it does not
exert any cytotoxicity even at high concentration (1, 13).
Compared with other known antioxidants, hydrogen has
many distinct advantages, such as selective oxidation,
non-toxicity, high permeability, no residues, relative
safety, and low cost (9).
Combining the main mechanism of the dogs' onion
poisoning with biological applications of hydrogen, the
purpose of this study was to assess the therapeutic effect
of hydrogen on the damages induced by onion poisoning
in dogs.
Material and Methods
Model establishment and sample collection.
A total of 16 adult beagle dogs were used in this
experiment. The dogs were vaccinated, individually
housed, and no substances that would affect the results
of the experiment were administered during the
experiment. All dogs were divided into two equal
groups: control group (group C) in which the dogs did
not undergo any treatment after poisoning, and hydrogen
group (group H2) in which the dogs were injected with
hydrogen after poisoning. All dogs were fed dehydrated
onion powder at the dose of 10 g/kg for three days.
Onion powder was mixed with cooked meat when fed.
Heinz's ratio of more than 50% and haematuria indicated
that the poisoning model had been successfully
established. Hydrogen was produced by hydrogen
generator (Saikesaisi, China) and was stored in a sealed
bag, using a syringe to extract hydrogen gas from the
exhaust hole when needed. The dogs in group H2 were
injected hydrogen subcutaneously in the dose of
0.2 mL/kg (23) for 12 days, in which the Heinz bodies
almost disappeared. Blood samples of two groups were
collected from the jugular vein one week before feeding
the onion powder (day –7), one day before injecting
hydrogen (day 0), and 2 h after injection of hydrogen gas
on days 1, 3, 5, 7, 9, and 12 of experimental treatment.
Index measurement and method. Indexes
measured included five aspects: clinical symptoms,
routine blood indexes, blood smears, liver and kidney
function, and antioxidant system of erythrocytes.
In the entire course of the experiment, the clinical
symptoms were closely observed, mainly including the
following aspects: urine colour, dietary status, body
temperature, and status of visible mucosa.
The haematology index was tested timely. Red
blood cell (RBC) and white blood cell (WBC) counts,
haemoglobin (HGB), and haematocrit (HCT) were
determined with a fully automated haematology analyser
(MEK-7222K, Japan).
Blood smears were mainly used to detect Heinz
bodies and reticulocytes. Fresh blood was stained with
Wright Giemsa staining method to count Heinz bodies,
which were determined by the percentage of cells that
had Heinz bodies within a population of 500
erythrocytes counted (7). Reticulocytes were observed
after staining with brilliant cresyl blue. The stain and
blood were mixed in a 1:1 ratio, incubated for 15 min or
longer at room temperature, then blood smears were
assessed according to conventional criteria. At least
1,000 RBCs were counted and Miller′s method was used
to make reticulocyte counting precise. The reticulocyte
cytoplasm contained light or dark blue mesh structure
after staining.
The anticoagulated blood was centrifuged at
2,500 g/min for 10 min, and the plasma was used to
determine the concentration of biochemical markers
characteristic for hepatic and renal functions, and
haemolytic ratio. Blood urea nitrogen (BUN), creatinine
(CRE), alanine aminotransferase (ALT), aspartate
aminotransferase (AST), total bilirubin (TBIL), and
direct bilirubin (DBIL) were determined. These
compounds were tested using an automated biochemical
analyser (IDEXX, USA).
After centrifuging the anticoagulated blood, the
precipitated RBCs were washed first and physiological
saline was added in a volume three times bigger than that
of RBCs, then the solution was centrifuged for 5 min,
and the RBCs pellet was washed again in physiological
saline. The process was repeated three times until the
supernatant was colourless. RBCs were extracted
quantitatively and cold distilled water was added
proportionately for hemolysate to measure malondialdehyde
(MDA), the total antioxidant capacity (T-AOC), reduced
glutathione (GSH), catalase (CAT), and the hydroxy
radical inhibition activity in the antioxidant system of
erythrocytes using reagent kits (Nanjing Jiancheng
Bioengineering Institute, China).
Statistical methods. Using SPSS software package
(Version 22.0) for statistical data analysis, the results
were shown as means ± standard deviation (± SD).
Independent-sample t-test was adopted to compare
results of two groups (group C and group H2) after the
test of normality and the assumption for homogeneity of
variances. P value <0.05 was considered statistically
significant.
Results
Clinical symptoms. All dogs were healthy and
active before feeding onions. After feeding onions for
three days, all dogs in groups C and H2 showed
haematuria, increased body temperature, and pale visual
mucosa. On the 3rd day after the injection of hydrogen,
the urine of five dogs in group H2 turned yellow, but
only two dogs in group C showed signs of improvement.
On the 10th day after injection of hydrogen, the clinical
symptoms of both groups returned to normal.
Routine blood indexes. WBC increased
dramatically after poisoning, and both groups reached
the peak on day 3. The counts of WBC in group H2 were
lower than those in group C. Significant differences were
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J. Zhao et al./J Vet Res/61 (2017) 527-533 529
noted on days 7 and 12 (P < 0.01) (Fig. 1A). RBCs in the
two groups decreased after poisoning and reached
minimum on day 3, then began to recover. The counts of
RBC in group H2 were higher than those in group C on
days 5, 9, 12 (P < 0.05), and on day 7 (P <0.01) (Fig.
1B). HGB declined sharply after poisoning and did not
recover to normal levels on day 12. HGB in group H2
was higher than in group C in the period of hydrogen
injection, and the difference was significant on days 7
and 12 (P < 0.05), and highly significant on day 9
(P < 0.01) (Fig. 1C). HCT decreased until day 3, then
increased slowly after poisoning, and in group H2 was
higher than in group C, particularly on day 5 (P < 0.01)
and on day 7 (P < 0.05) (Fig. 1D).
Blood smear examinations and haemolytic ratio.
Heinz bodies in group H2 disappeared completely first
and were lower than in group C, and a significant
difference was observed on days 5, 7 (P < 0.05), and 9
(P < 0.01) (Fig. 2A). Reticulocyte counts in group H2
were higher than in group C, especially on days 3
(P < 0.05), 5, and 7 (P < 0.01) (Fig. 2B). The haemolytic
ratio counts increased first and then decreased in the two
groups, and both reached the peak on day 3. The counts
in group H2 were lower than in group C, particularly on
days 3, 9, and 12 (P < 0.01) (Fig. 2C).
Liver and renal function measurements. The
ALT in group H2 was lower than in group C during
hydrogen injection and showed significant difference on
day 7 (P < 0.01). The AST in group H2 was lower than in
group C on days 1, 3, 5, and 7 and there was a significant
difference on day 5 (P < 0.01). The values of TBIL and
DBIL had similar trends and TBIL values in group H2
were lower than in group C (except on days 1 and 12),
particularly on day 5 (P < 0.01). The changing trends of
BUN and CRE were irregular with fluctuations in the
normal range. There was no significant difference
between the two groups at other time points except on
day 9 for CRE (P < 0.05) (Fig. 3 A-F).
Antioxidant system of erythrocytes. The MDA
level in group H2 were lower than in group C (except day 1)
and significant difference was noted on days 9 (P < 0.05),
5, and 7 (P < 0.01). The changing trend for T-AOC after
poisoning was irregular. T-AOC values in group H2 were
higher than in group C (except day 1), especially on days
3, 9 (P < 0.01), and 12 (P < 0.05). The hydroxy radical
inhibition activity declined sharply after poisoning, and
the values in group H2 were higher than in group C from
the third day after injecting hydrogen, with significant
difference observed on day 7 (P < 0.05) and on days
3 and 9 (P < 0.01). The values of CAT did not have
a regular changing trend and there was no significant
difference between the two groups. The concentration of
GSH in the two groups decreased rapidly after poisoning
and reached the minimum on day 3; then it began to
recover. The values in group H2 reached the maximum
on day 7, then decreased and fluctuated as the values
before poisoning. The values in group H2 were higher
than in group C on days 5 and 7 (P < 0.01). Detailed
results are shown in Table 1.
Fig. 1. Comparison of the values of white blood cells (WBCs), red blood cells (RBCs), haemoglobin (HGB), and haematocrit
(HCT) between the control group (group C) and hydrogen group (group H2) on day 7 before injecting hydrogen, on day 0 (one
day before injecting hydrogen), and on days 1, 3, 5, 7, 9, and 12 during the injection of hydrogen. * P < 0.05, ** P < 0.01
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530 J. Zhao et al./J Vet Res/61 (2017) 527-533
Fig. 2. Comparison of counts of Heinz bodies, reticulocytes, and haemolytic ratio between the control group (group C)
and hydrogen group (group H2) on day 7 before injecting hydrogen, day 0 (one day before injecting hydrogen), and on
days 1, 3, 5, 7, 9, and 12 during the injection of hydrogen. * P < 0.05, ** P < 0.01
Table 1. The erythrocyte antioxidant system results (mean ± SD) in the control group (group C) and hydrogen group (group H2) on day 7 before
injecting hydrogen, on day 0 (one day before injecting hydrogen), and on days 1, 3, 5, 7, 9, and 12 during the injection of hydrogen gas
Time
Group
MDA (nmol/mL）
T-AOC (U/mL RBC)
·OH IA（U/mgHb）
CAT (U/mgHb)
GSH (μmol/L)
Day (–7)
Day 0
Day 1
Day 3
Day 5
Day 7
Day 9
Day 12
Group C
Group H2
Group C
Group H2
Group C
Group H2
Group C
Group H2
Group C
Group H2
Group C
Group H2
Group C
Group H2
Group C
Group H2
4.5 ± 0.98
4.27 ± 0.92
9.48 ± 1.27
8.69 ± 0.98
16.79 ± 1.67
17.41 ± 0.7
16.48 ± 2.16
16.0 ± 2.87
9.6 ± 1.66
7.19 ± 1.34**
6.94 ± 1.2
5.03 ± 1.18**
4.54 ± 0.57
3.73 ± 0.86*
2.77 ± 0.42
2.47 ± 0.15
324.63 ± 28.03
318.08 ± 21.87
279.95 ± 25.55
281.2 ± 22.81
232.12 ± 20.52
231.21 ± 27.33
240.47 ± 21.2
299.08 ± 12.82**
256.98 ± 26.59
274.56 ± 31.59
304.76 ± 35.84
313.13 ± 24.93
247.79 ± 24.56
345.56 ± 37.97**
270.88 ± 22.56
304.01 ± 30.42*
89.73 ± 8.35
84.12 ± 8.89
55.91 ± 11.68
53.40 ± 11.96
38.06 ± 6.79
34.8 ± 5.9
22.36 ± 3.66
27.28 ± 2.56**
53.66 ± 6.57
55.82 ± 8.38
65.43 ± 8.38
73.87 ± 6.98*
71.1 ± 6.98
82.3 ± 6.46**
79.81 ± 11.41
77.07 ± 12.7
5.05 ± 1.15
4.81 ± 1.11
2.88 ± 0.54
2.83 ± 0.48
4.04 ± 1.22
3.89 ± 0.81
3.64 ± 1.23
4.68 ± 1.06
5.38 ± 1.16
6.37 ± 1.1
6.34 ± 1.62
5.69 ± 1.36
6.45 ± 1.61
7.14 ± 0.91
7.27 ± 1.71
7.76 ± 0.71
166.09 ± 16.46
165.56 ± 12.95
133.63 ± 9.63
125.68 ± 9.11
93.44 ± 13.77
78.59 ± 11.37*
50.35 ± 8.42
56.35 ± 6.0
104.53 ± 8.08
122.68 ± 6.77**
143.91 ± 9.86
201.07 ± 14.3**
171.8 ± 12.01
164.64 ± 12.84
184.3 ± 20.66
170.37 ± 7.31
MDA – malondialdehyde; T-AOC – total antioxidant capacity; ·OH IA – hydroxy radical inhibition activity; CAT – catalase; GSH – reduced
glutathione. * P < 0.05, ** P < 0.01
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J. Zhao et al./J Vet Res/61 (2017) 527-533 531
Fig. 3. Comparison of the values of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total
bilirubin (TBIL), direct bilirubin (DBIL), blood urea nitrogen (BUN), and creatinine (CRE) between the control
group (group C) and hydrogen group (group H2) on day 7 before injecting hydrogen, on day 0 (one day before
injecting hydrogen), and on days 1, 3, 5, 7, 9 and 12 during the injection of hydrogen. * P < 0.05, ** P < 0.01
Discussion
In this study, the model of onion poisoning in dogs
was successfully established, and typical haemolytic
anaemia symptoms were observed in the animals.
A previous study (11) has revealed that onion
poisoning occurs in dogs when the amount of onions
fed accounts for more than 0.5% of the animal’s
weight. Considering the different moisture content of
fresh onions, dehydrated onion powder was used in this
study because of ease of administration and better
control of the dose (7). The final dose of onion powder
was based on the results of a preliminary study.
The routine blood indexes, blood smear,
biochemical markers of liver and kidney function, and
erythrocyte antioxidant system all basically followed
the trend of poisoning-recovering. Heinz bodies and
MDA increased to peak and then decreased, which was
consistent with the results of previous research (24).
Some physiological indicators suggested that the dogs
in group H2 recovered first in relation to group C, e.g.
Heinz bodies which disappeared first in the group H2.
However, all dogs in two groups returned to healthy
physiological state by the end of experiment. The dogs
in group C were also self-healing finally due to the
effective removal system of RBC, which included two
sets of antioxidant systems. One was enzymatic system,
such as superoxide dismutase (SOD), and the other
was non-enzymatic system including vitamin C,
glutathione, vitamin E, etc. (4, 16). These specific
scavenging systems weaken the toxic effects of
hydrogen peroxide and other ROS (2), and eventually
relieved the symptoms of poisoning in the dogs which
recovered in the end.
There was no drastic change in CRE and BUN in
comparison to the period before feeding onions. It was
because the kidneys had a strong reserve and
compensatory capacity, so CRE and BUN were usually
at the healthy level in the early detection of renal
damage, until substantial impairment of the kidneys
was observed or the development of end-stage renal
disease occurred. The results indicated that the
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532 J. Zhao et al./J Vet Res/61 (2017) 527-533
poisoned dogs did not show severe kidney damage in
the experimental process, which corresponded to the
previous study stating that serum BUN or UC ratio and
glomerular filtration rates (GFR) did not show an
increase in anaemic dogs (15).
Hydrogen influenced the selective oxidation and
could react with the active oxygen, such as •OH and
ONOO-, that had very strong oxidation directly (20).
The results obtained demonstrated that the levels of
hydroxy radical inhibition activity in group H2 were
significantly higher than in group C from the third day
of hydrogen injection, and dogs of the group H2
recovered first. T-AOC, GSH, CAT, and MDA in
group H2 showed significant differences on some days
after hydrogen injection, suggesting that the hydrogen
could improve the antioxidant levels of the body and
lower the levels of oxidative products, which was also
shown in previous studies (3, 27). This was not proven
to be the direct effect of hydrogen, but it might be
related to the mutual relation between the oxidation
system, as high level of intracellular GSH could cause
reduction reaction with H2O2, which had a direct
correlation with CAT.
The counts of reticulocytes in group H2 were
significantly higher than in group C, indicating that
hydrogen could improve bone marrow haematopoietic
capacity (12), and the trend was consistent with the
changes of RBC. MDA was one of the lipid
peroxidation products. Hydrogen peroxide and MDA
could oxidise the sulfhydryl groups of haemoglobin,
causing the denaturation of haemoglobin and the
formation of Heinz bodies (19, 24). The number of
Heinz bodies between the two groups was in
conformity with MDA change.
The lipid peroxidation also decreased the fluidity
and deformation of erythrocyte membrane (24),
resulting in more serious damage of erythrocytes,
which would cause haemoglobinaemia and
haemoglobinuria. The values of the haemolytic rate,
which had positive correlation with the injury degree of
erythrocyte membrane, were lower in group H2 than in
group C on some days, showing that hydrogen could
inhibit the lipid peroxidation at a certain extent, reduce
the oxidative damage of erythrocyte membrane, leading
to reduction of the haemolysis degree in the poisoned
dogs. The significant differences in the blood indexes
observed in the present study between the two groups
indicate that hydrogen could reduce the oxidative
damage of the erythrocytes, and then lead to reduction
in the degree of haemolytic anaemia.
In conclusion, the results indicate that continuous
injection of hydrogen in onion poisoned dogs would
relieve haemolytic anaemia and increase bone marrow
haematopoietic ability. Hydrogen could also reduce the
oxidative damage of erythrocyte membrane by
reducing oxidation products and increasing antioxidant
substances, thus causing reduction of the number of
Heinz bodies and haemolysis rate. Hydrogen could be
considered as an auxiliary treatment drug for onion
poisoned dogs in the future.
Conflict of Interests Statement: The authors declare
that there is no conflict of interests regarding the
publication of this article.
Financial Disclosure Statement: This study was
funded by the National Natural Science Foundation of
China, China Postdoctoral Science Foundation, and
Young Talents Project of Northeast Agricultural
University.
Animal Rights Statement: All animal procedures
were approved by the Laboratory Animal Care and Use
Committee of Northeast Agricultural University.
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