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Arsenic-induced testicular toxicity in Gallus gallus: Expressions

of inflammatory cytokines and heat shock proteins

Xiao Sun,2 Siwen Li,2 Ying He, Hongjing Zhao, Yu Wang, Xiangwei Zeng,1 and Mingwei Xing1

College of Wildlife Resources, Northeast Forestry University, Harbin 150040, Heilongjiang Province, China

ABSTRACT The aim of the study is to investigate Hsp60, Hsp40, and Hsp27 were assessed by quantita-
the effects of sub-chronic poisoning with arsenic on tive real-time PCR in the testes of chickens. The protein
the testes of chickens treated. Seventy-two 1-day-old expressions of iNOS, Hsp60, and Hsp70 were detected
chickens were randomly divided into 4 groups and pro- by western blot. Increased mRNA and protein levels of
vided food with different doses arsenic. The histological inflammatory factors and Hsps with testicular damage
changes were examined. The mRNA levels of inflam- showed that arsenic-induced testicular toxicity includes
matory factors, including transcription factor nuclear inflammatory and heat shock response in chickens, and
factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), that increased Hsps levels may play a protective role in
inducible nitric oxide synthase (iNOS), cyclooxygenase- inflammation damage induced by arsenic on the testes
2 (COX-2), prostaglandin E synthase (PTGEs), and of chickens.
heat shock proteins (Hsps), including Hsp70, Hsp90,
Key words: Arsenic, Chicken, Testicle, Inflammatory factor, Heat shock protein
2017 Poultry Science 0:1–8

INTRODUCTION clear factor-κB (NF-κB) can regulate the expressions

of okadaic acid, a phosphatase 1 and 2A inhibitor in-
Arsenic is one of the most common toxins and a ma- volved in the inflammatory response (Schmidt et al.,
jor health concern globally due to its wide distribution 1995). After activation of NF-κB, it can regulate the
and adverse health effects. It is widely used as a feed expression of a series of genes, including tumor necrosis
additive in the production of animals at present. Ar- factor-α (TNF-α) and inducible nitric oxide synthase
senic has been proposed as a cause of reproductive (iNOS), among others (Schmidt et al., 1995). TNF-α
failure in male workers at a copper smelter in Swe- is an important pro-inflammatory cytokine mainly pro-
den, but this suggestion should be viewed with cau- duced by a series of immune cells, including T lympho-
tion, as subjects were also exposed to other potentially cytes, B lymphocytes, natural killer cells, eosinophils,
hazardous metals (Beckman, 1978). Nevertheless, ar- basophils, neutrophils, and monocytes (Varfolomeev
senic is considered to be a likely reproductive toxicant and Ashkenazi, 2004). It can also be generated by some
that might cause male-mediated reproductive effects non-immune cells, including Kupffer cells, fibroblasts,
(Hopenhayn-Rich et al., 1999). In the epididymides smooth muscle cells, endothelial cells, glial cells, and
of mice, the presence of radioactive arsenic indicated neurons (Thomson and Lotze, 2003). Moreover, TNF-
a risk of decreasing sperm viability and impaired α can produce calcium-independent iNOS, release NO,
reproduction (Danielson et al., 1984; Pant et al., 2001). and cause tissue inflammation (Blanco et al., 2007). En-
Furthermore, it was reported that arsenic exposure pro- zymes of iNOS and cyclooxygenase-2 (COX-2), which
duced steroidogenic dysfunction causing impairment of are involved in tissue damage, can induce prostaglandin
spermatogenesis in rats (Sarkar et al., 2003). E synthase (PTGEs; Surh et al., 2001). Therefore, re-
Arsenic exposure influences the expression of inflam- search on these inflammatory cytokines in the testes
matory cytokines in the airway of women (Dutta et al., of chickens treated with arsenic trioxide (As2 O3 ) may
2015). Inflammation is a defensive reaction to injury be advantageous to understand whether heavy metal
factors of the vascular system in living tissue. Many arsenic induces an inflammatory response.
inflammatory cytokines are involved in the inflamma- Inflammatory cytokines can induce heat shock pro-
tory response: for instance, the transcription factor nu- teins (Hsps) upon infection with a virus (Phillips et al.,
1991). In 1962, Ritossa observed that the polytene chro-

C 2017 Poultry Science Association Inc. mosomes of normal flies showed “puffing”; later, the
Received November 30, 2016. transcription of those puffs were strengthened under
Accepted March 15, 2017.
Corresponding author:
high temperature, which may prompt some kind of Hsps
Xiao Sun and Siwen Li contributed equally to this work. synthesis (Tissieres et al., 1974; Ritossa, 1996). Hsps are


a group of proteins that are generated by environmen- with sodium pentobarbital (National Research Council,
tal stressors under high temperatures (Del et al., 2001). 1994).
Hsps are highly conserved during the process of evolu-
tion, such that the Hsps homology of bacteria and hu-
mans shares more than 50% (Craig, 1985). Among these Euthanasia and Testes Collection
proteins, Hsp70 is the most important Hsps (Hunt and Animals were anesthetized and euthanized by injec-
Morimoto, 1985). To avoid aggregation, Hsp70 is able tion of sodium pentobarbital (30 mg/kg body weight),
to recognize and bind to hydrophobic groups around and the testes of the chickens removed quickly on
them before folding the precursor protein. Its function days 30, 60, and 90, respectively. Then the testis tis-
is to remit cell injuries and enhance the body’s resis- sue specimens were fixed with 2.5% glutaraldehyde in
tance to diverse stressors (Kang et al., 1990). Hsp90 0.1 M sodium phosphate buffer for electron microscopy,
is an important molecular chaperone that participates and the other samples were directly stored at −80◦ C
in the synthesis of many key molecules within the cell until further use.
(especially those regulating cell growth and the sur-
vival of many signaling molecules), affecting the sta-
bility and function of these molecules (Neckers and Ivy, Ultrastructural Observations
2003). Hsp60 synthesis is stimulated by protein denatu-
For electron microscopy, testis tissue specimens were
ration (Hightower, 1993). Hsp40 and Hsp27 are mainly
fixed with 2.5% glutaraldehyde in 0.1 M sodium phos-
involved in the actin stability and cytokine signal trans-
phate buffer (pH 7.2) for 3 h at 4◦ C, washed in the
duction, protecting cells from damage of various stress
same buffer for 1 h at 4◦ C and post-fixed with 1% os-
factors (Mehlen et al., 1996).
mium tetroxide in sodium phosphate buffer for 1 h at
To the best of our knowledge, few studies have ex-
4◦ C. The tissues were then dehydrated in a graded se-
amined the expression of inflammatory cytokines and
ries of ethanol starting at 50% ethanol for 10 min at a
heat shock proteins in relation to arsenic-induced tes-
time and then were immersed twice in propylene oxide.
ticular toxicity. The present study was performed to
The tissue specimens were mounted using a 10:1 ratio
investigate the protective effects of inflammatory cy-
of epoxy (Araldite GY-502 Epoxy, Huntsman Interna-
tokines and heat shock proteins on testicular toxicity
tional LLC, Salt Lake City, UT): hardener (Hysol HD
using chicken as a model.
3416 Hardener, Loctite/Henkel Corp., Atlanta, GA).
Ultrathin sections were stained with Mg-uranyl ac-
etate and lead citrate for evaluation, and testis samples
MATERIALS AND METHODS were observed by a transmission electron microscope
(Xing et al., 2015).
Animal Care and Drug Treatment
All procedures used in this study were approved by The mRNA Levels of Inflammatory
the Institutional Animal Care and Use Committee of Cytokines and Hsps in the Testes
Northeast Forestry University (Harbin, China) (UT-31; of Chickens Exposure to As2 O3
20 June 2014). The highest dose of sub-chronic tox-
icity test can use 1/20 to 1/5 of the median lethal Specific primers for inflammatory cytokines (NF-κB,
dose (LD50 ) [Appendix 1], and LD50 of arsenic for TNF-α, iNOS, COX-2, PTGEs), Hsps (Hsp70, Hsp90,
chicken was 50 mg/kg BW [Appendix 2]. Base on that, Hsp27, Hsp40, Hsp60), and the internal reference of
72 1-day-old chickens were randomly divided into 4 glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
groups: the low arsenic dose group (L group, contain- were designed by the Primer Premier software (PRE-
ing 7.5 mg/kg As2 O3 ), the medium arsenic dose group MIER Biosoft International, Palo Alto, CA) referring
(M group, containing 15 mg/kg As2 O3 ), the high to known chicken sequences (Appendix 3).
arsenic dose group (H group, containing 30 mg/kg Total RNA was extracted from the testes (50 mg tis-
As2 O3 ), and the control group (C group, containing sue, n = 6/group) using RNAiso Plus reagent (Takara,
0 mg/kg As2 O3 ). As2 O3 was added into the food to China). The concentration and purity of the total
make supplements uniformed according to the chicken RNA were determined spectrophotometrically at the
LD50 of As2 O3 . Each group contained 18 chickens. The absorbance of OD260/280 nm (Gene Quant 1300/100,
experimental animals were provided adequate food and USA). The RNA was reverse-transcribed to cDNA us-
water. Each chicken was fed with the same amount of ing the PrimeScript RT Reagent Kit (Takara, China)
feed, and detected the changes of body weight. according to the manufacturer’s instructions. Synthe-
The composition of the diet was 2.766% metabo- sized cDNA was diluted 10 times with sterile, double-
lizable energy (kcal/kg), 18.45% crude protein, 2.82% distilled water and stored at −80◦ C before use.
crude fiber, 3.80% calcium, and 0.62% phosphorus, Quantitative real-time PCR was completed using
which had been added to the feedstuff to meet the the FastStart Universal SYBR Green Master reagents
minimum requirements for energy and nutrients for (Roche USA, Branford CT) on a BIOER LineGene
chickens and without influencing subsequent euthanasia 9600 sequence detection system (BIOER, China). Each

Figure 1. Ultrastructural changes of chickens’ testes tissue sections. A and F represented testes of chickens from Control group. B and G
represented testes of chickens from L groups. C and H represented testes of chickens from M group. D, E, and I represented testes of chickens
from H groups at 90 days.

sample was evaluated in triplicate. The detailed con- Statistical Analyses

ditions of PCR protocol and the method of the rela-
tive mRNA abundance on the basis of the method of All statistical analyses of the data were performed
2−ΔΔCt were indicated in our previous research (Leite using SPSS 13.0 software. The level of statistical about
et al., 2013; Guo et al., 2016). significant differences value (P < 0.05) was obtained by
one-way analysis of variance (ANOVA). Data regard-
ing differences between the mean values of normally dis-
Western Blot Analysis of iNOS, Hsp60, tributed data were assessed by Tukey’s honestly signif-
icant difference test for post hoc multiple comparisons.
and Hsp70 Data were expressed as the mean ± SD of 6 observa-
All of the 50 mg of testis tissue for each sample were tions.
rinsed in saline and then homogenized in sodium do-
decyl sulfate (SDS) Lysis Buffer (Beyotime, China),
which contained 1 mM phenylmethylsulfonyl fluoride.
After centrifuging, the supernatants were collected. The Ultrastructural Changes
concentrations of protein were detected using BCA pro-
tein assay kits to equal the protein extracts doses of all Testes tissues from the control group showed nor-
groups. mal ultrastructure structures with regular cells on
The protein extracts were separated by 12% SDS- 90 days (Figure 1A and 1F). In the corresponding L,
polyacrylamide gel electrophoresis. The separated pro- M, H group, arsenic treatment caused extensive injury
teins were transferred to a polyvinylidene fluoride of the testis tissues. The mitochondria in testis tissues
(PVDF) membrane, then blocked for 2 h at 37◦ C of the arsenic groups were swollen and vacuolated with
and incubated with rabbit anti-chicken iNOS, Hsp60 or degeneration or loss of cristae. Cells showed typical
Hsp70 polyclonal antibodies (1:1000, provided by Prof. chromatin condensation and margination and shrink-
Xu, Northeast Agricultural University), and GAPDH age of their nuclei comparison with the control group
antibody (1:1000, Beyotime, China) overnight at 4◦ C. (Figure 1B-E and 1G-I), and there was apoptosis with
After being washed 3 times with Tris-buffered NaCl perinuclear chromatin condensation in H group. The
solution with tween 20 (TBST), the membrane was alteration degree was in a dose-dependent manner.
then incubated with a horseradish peroxidase (HRP)-
labeled goat anti-rabbit IgG (1:10000, Beijing Biosyn-
thesis Biotechnology Co., LTD, China) for 1 h at 37◦ C. Effects of As2 O3 on the mRNA Levels
By using the enhanced chemiluminescence (ECL) west- of NF-κB, TNF-α, iNOS, COX-2, and PTGEs
ern blotting detection kit (Thermo Scientific, Waltham, in the Testes of Chickens
MA), the protein expression bands were visualized, and
then the membrane was exposed to an X-ray film. The The effects of As2 O3 on the mRNA levels of in-
GAPDH signal was used as an internal reference pro- flammatory cytokines in testes of chickens were shown
tein standardization to obtain relative amounts of the in Figure 2. The NF-κB and TNF-α mRNA levels of
proteins of each sample. testes tissues were significantly increased (P < 0.05 or

Figure 2. Effects of As2 O3 on the mRNA levels of NF-κ B, TNF-α , iNOS, COX-2, and PTGEs in the testes of chickens. A represented the
mRNA levels of NF-κ B in the testes. B represented the mRNA levels of TNF-α in the testes. C represented the mRNA levels of iNOS in the
testes. D represented the mRNA levels of COX-2 in the testes. E represented the mRNA levels of PTGEs in the testes. Each value represented
the mean ± SD of 6 individuals (n = 6). The asterisk or double asterisk indicates that there were significant differences (∗ P < 0.05 or ∗∗ P <
0.01) between the C group and L, M, H group at the same time point, respectively.

P < 0.01) in arsenic treatment groups compared with Hsp40, and Hsp27 mRNA transcription increased in a
control groups, except for the L group on days 30 with dose-dependent manner (P < 0.05 or P < 0.01).
no significant changes (Figure 2A and B). Compared
with control groups, the iNOS, COX-2, and PTGEs
mRNA levels of testes tissues were significantly in- Western Blot Analysis of iNOS Protein
creased (P < 0.05 or P < 0.01) (Figure 2A-E). Expressions in the Testes of Chickens
In order to investigate the effects of arsenic-toxicity
on inflammation-related protein expressions in testes
Effects of As2 O3 on the mRNA Levels of chickens, iNOS expressions were examined by West-
of Hsp70, Hsp90, Hsp60, Hsp40, and Hsp27 ern blot and showed in Figure 4. Compared with the
in the Testes of Chickens corresponding control groups, the protein expressions
of iNOS in the chronic arsenic treatment group for
Effects of As2 O3 on the mRNA levels of Hsp70, iNOS increased significantly (P < 0.05 or P < 0.01)
Hsp90, Hsp60, Hsp40, and Hsp27 in testes of chickens (Figure 4B).
were shown in Figure 3. The mRNA levels of Hsp70,
Hsp90, Hsp40, and Hsp27 in the testes were shown in
Figure 3A, B, D and E. The L, M, and H groups on Western Blot Analysis of Hsp60 and Hsp70
days 30, 60, and 90 had a significant increase (P < 0.05 Protein Expressions in the Testes of
or P < 0.01) compared with those in the correspond- Chickens
ing control groups. The mRNA levels of Hsp60 in the
testes were shown in Figure 3C. Compared with the In order to investigate the effects of arsenic-toxicity
control group, the M and H group on days 30, 60, and on Hsps levels in testes of chickens, Hsp60 and Hsp70
90 had a significantly increase (P < 0.05 or P < 0.01). expressions were examined by Western blots. The pro-
From the information above, the Hsp70, Hsp90, Hsp60, tein expressions of the H groups for Hsp60 in the testes

Figure 3. Effects of As2 O3 on the mRNA levels of Hsps in the testes of chickens. A represented the mRNA levels of Hsp70 in the testes. B
represented the mRNA levels of Hsp90 in the testes. C represented the mRNA levels of Hsp60 in the testes. D represented the mRNA levels of
Hsp40 in the testes. E represented the mRNA levels of Hsp27 in the testes. Each value represented the mean ± SD of 6 individuals (n = 6). The
asterisk or double asterisk indicates that there were significant differences (∗ P < 0.05 or ∗∗ P < 0.01) between the C group and L, M, H group at
the same time point, respectively.

in all groups increased significantly on days 30 and 90 cell infiltration, typical chromatin condensation and
(P < 0.05 or P < 0.01) in comparison to the correspond- margination, and shrinkage of their nuclei in compari-
ing control groups (Figure 5B). However, the protein son with the control group. Results indicated that the
expressions of the H group for Hsp60 were decreased tissues of chickens treated with arsenic were damaged
significantly on days 60 (P < 0.05 or P < 0.01) (Fig- in a time- and dose-dependent manner compared with
ure 5B). Nevertheless, the protein expressions of the the control group. We found arsenic ingestion reduced
H group for Hsp70 was increased significantly in com- chicken body weight slightly; however, the testes weight
parison to the control (P < 0.05 or P < 0.01) (Fig- tended to be reduced significantly (Appendix 5, unpub-
ure 5C), except for the H group on days 60 with no lished data), which was similar with Adedara’s study
significant changes. (Adedara et al., 2017). In addition, the heart induced
by chronic arsenic toxicity was reported to be partially
DISCUSSION reversible after cessation of As2 O3 administration (Wu
et al., 2003).
Arsenic, a heavy metal, is toxic when it penetrates We examined the inflammatory reactions, such as the
into human and animal bodies by inhalation (Dutta mRNA levels of NF-κB, TNF-α, iNOS, COX-2, and
et al., 2015). Following arsenic exposure, the matu- PTGEs on the testes by quantitative real-time PCR
ration of spermatogonia through the process of meio- (qPCR). Based on our results, we observed that the
sis has been reported to be severely disrupted (Omura NF-κB, TNF-α, iNOS increased in a time- and dose-
et al., 2000; Sanghamitra et al., 2007). Tissues are dam- dependent manner. COX-2 and PTGEs mRNA levels
aged following the change of the expression of inflam- of testes tissue were significantly increased, suggest-
matory cytokines and heat shock proteins (Zhao et al., ing that both of them may play influential roles in
2013). For the purpose of detecting arsenic-induced tis- tissue inflammation injury induced by arsenic toxic-
sues damage, we examined the histological (Appendix ity within the testes. Previous studies have reported
4, unpublished data) and ultrastructural observations, the similar findings that arsenic toxicity could disrupt
which showed testicular damage with inflammatory the homeostasis and cause inflammation injury to the

gastrointestinal tract of chickens (Xing et al., 2015). In

addition, lead induced inflammatory damage in testes
of Swiss albino mice (Barbhuiya et al., 2013), and
the mRNA level of TNF-α in the zebrafish larvae
treated with copper increased in a time-dependent man-
ner (Leite et al., 2013). Results were consistent with
the previous conclusion that PTGEs catalyzed the iso-
merization of PGH2 to PGE2, and the latter played
an important role in inflammation (Liu et al., 2012),
cell growth, and transformation (Martin et al., 1991).
TNF-α induces the activation of NF-κB in the COX-2
promoter, followed by initiation of COX-2 expression
(Chen et al., 2000). Our results were similar to a prior
study by Fu et al. (2013), which showed that expression
of COX-2 and PTGEs in the intestinal tract were in-
creased by cold stress at early time points, but reduced
after 20 d of cold exposure. Our results showed that
after adding As2 O3 to the food, COX-2 mRNA levels
in M and H groups increased on days 30 and 60 com-
pared with the corresponding control groups; however,
Figure 4. Effects of As2 O3 on proteins expressions of iNOS in the these declined on d 90, so we surmised that TNF-α in-
testes of chickens. A represented the protein expressions of iNOS in
the testes. C30 , C60 , and C9 0 represented C groups at 30, 60, and duced the activation of COX-2. Compared with control
90 d, respectively. H30 , H60 , and H90 represented H groups at 30, 60, group, our results revealed that excess arsenic could up-
and 90 d, respectively. B represented iNOS/GAPDH ratio in the testes. regulate NF-κB, TNF-α, iNOS, COX-2, and PTGEs
The values presented the means ± SD of three individuals (n = 3). mRNA levels.
The asterisk or double asterisk indicates that there were significant
differences (∗ P < 0.05 or ∗∗ P < 0.01) between the C group and H The mRNA expression levels of Hsps were up-
group at the same time point, respectively. regulated compared with control groups; as molecular
chaperones, Hsps are synthesized by a group of spe-

Figure 5. Effects of As2 O3 on the protein expressions of Hsp60, Hsp70 in the testes of chickens. A represented the protein expressions of
Hsp60, Hsp70 and GAPDH in the testes. C30 , C60 , and C90 represented C groups at 30, 60, and 90 d, respectively. H30 , H60 , and H90 represented
H groups at 30, 60, and 90 d, respectively. B represented Hsp60/GAPDH ratio in the testes. C represented Hsp70/GAPDH ratio in the testes.
The values presented the means ± SD of three individuals (n = 3). The asterisk or double asterisk indicates that there were significant differences
(∗ P < 0.05 or ∗∗ P < 0.01) between the C group and H group at the same time point, respectively.
cific protein when under stress, such as high fever, rate, suggesting that PTGEs might be inhibited by
oxidative stress, infections, nutritional deficiencies, or Hsps. This is consistent with our results.
exposure to harmful chemicals and inflammatory cy- Arsenic causes development of various hazardous ef-
tokines (Lindquist and Craig, 1988; Yao et al., 2013a,b). fects on testes of chickens and induces testicular toxi-
In our study, the increased inflammation factors sug- city. Inflammatory and heat shock response were trig-
gested that an inflammatory response in the testes was gered and might play protective roles in inflammation
induced by As2 O3 , which might lead to testicular in- damage. Results of the present study strongly suggested
jury. Increasing numbers of research studies in the lit- that exposure to arsenic might have adverse effects on
erature support the important roles of Hsps in the in- testicular development. However, so far, the specific
flammatory response. Hsps are involved in signal trans- As2 O3 -activated induction and regulation mechanisms
duction and controlling of cytokine genes to enhance among Hsps and inflammatory cytokines are unclear,
the role of T lymphocyte antigen presentation, partic- and further studies are needed.
ipating in cell proliferation and the mechanism caused
by inflammation (Suzuki et al., 1991). Previous studies
have shown that As2 O3 increased mRNA expressions CONCLUSION
of Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90 in chicken In conclusion, the present experiments showed that
immune organs (Guo et al., 2016). Moreover, arsenite arsenic induced testicular toxicity in chickens. Arsenic
could stimulate Hsp70 expression in salmon (Grosvik exposure caused inflammatory and heat shock response,
and Goksoyr, 1996). Cao et al. reported that heavy and the increased of Hsps levels may play a protec-
metal (Mo, Ca) exposure raised the mRNA expressions tive role in inflammation damage induced by As2 O3 in
of Hsp60 and Hsp70, and there was a positive correla- chicken testes.
tion between metal concentration and expressions (Cao
et al., 2016). Thus, in our study, the increased inflam-
mation factors suggested that the testes inflammatory ACKNOWLEDGMENTS
response was induced by As2 O3 , which might lead to
This study was supported by the National Natural
testicular injury. Quite notably, in this study, mRNA
Science Foundation of China (Grant No. 31672619),
expression levels of Hsps were up-regulated comparing
the Fundamental Research Funds for the Central Uni-
with control groups, indicating that Hsps in testes were
versities (Grant No. 2572016EAJ5) and the Natural
overexpressed because of stress caused by heavy met-
Science Foundation of Heilongjiang Province (Grant
als and were synthesized in large quantities to protect
No. C2015061).
interstitial cell from damage through the Hsp path-
way. These results were also consistent with the study Conflict of interest: No author has any financial or per-
by Rajeshkumar and Munuswamy (2011), which re- sonal relationships that could inappropriately influence
ported that heavy metals such as Ag, Cd, and Cu could or bias the dose of the paper.
increase the Hsps expression in milk fish.
Expression of genes in the Hsp70 family may be
related to changes in pro-inflammatory cytokines in- SUPPLEMENTARY DATA
duced by exposure to As2 O3 (Sreedhar and Csermely, Supplementary data are available at PSCIEN online.
2004). By activation of phospholipase A2 (PLA2) and
promotion the rapid increase in intracellular ROS,
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