Apoptosis induced by acrylamide is suppressed in a 21.5% fat diet through caspase-3-independent pathway in mice testis.

Abstract

This study investigates the simultaneous effect of acrylamide (ACR) and high-fat-intake on the apoptosis in testis cells, and also the expression and activity of caspase-3. Seventy-two male Kunming mice were divided into two blocks and fed with a high-fat diet (crude fat 21.5%) or basic diet (crude fat 4.4%), respectively; and animals in each diet block were exposed to ACR at the dose of 20 mg/kgbw x d or 40 mg/kgbw x d as ACR treated groups or the normal saline as control. Germ cells prepared from testis were stained with Hoechst dye 33258 and paraffin wax sections from testis were suffered to a TUNEL process. Expression of caspase-3 on protein level was investigated using an immunohistochemical analysis assay. The supernatant of unilateral testes were subjected to a Caspase-3 activity kit to determine the activity of Caspase-3 in testis. The concentration of ACR and glycidamide(GA), epoxide of ACR, in plasma and testis were detected by LC-ES/MS/MS analysis. Results based on the morphological changes, percentage of apoptotic cells, and integrated optical density (IOD) of positive amethyst staining which indicates the apoptotic DNA fragmentation, show that apoptosis was induced by acrylamide only; however, acrylamide-induced apoptosis was weakened by high-fat-intake. The protein expression and activity of Caspase-3 were not induced by ACR or high-fat-intake. Moreover, no significant differences of ACR and GA concentration were found between the high-fat and basic diet groups after exposure of ACR. Results indicate that high-fat-intake reverses the effects on apoptosis induced by ACR; and more possibly, apoptosis is induced by a caspase-3-independent mechanism.

Full text

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Toxicology Mechanisms and Methods, 2009; 19(3): 219–224
ReseaRch aRticle
Apoptosis induced by acrylamide is suppressed
in a 21.5% fat diet through caspase-3-independent
pathway in mice testis
Xichun Zhang, Fahe Chen, and Zhiyong Huang
Bio-tech Engineering College, Jimei University, Xiamen, Fujian, PR China
Project supported by the Foundation for Innovative Research Team of Jimei University (No. 2006A003) and the Natural Science Foundation of Fujian Province of
China (No. 2007J0107).
Address for Correspondence: Zhang Xichun, Bio-tech College, Jimei University, Xiamen, Fujian, PR China 361021. Tel: 13400718894. E-mail: xczhang@jmu.edu.cn
(Received 9 August 2008; revised 19 September 2008; accepted 23 September 2008)
Introduction
In 2002, researchers demonstrated relatively high level of
acrylamide (ACR) in foods cooked at high temperature,
especially in carbohydrate-rich foods (Tareke et al. 2002).
These widely publicized findings stimulated worldwide
studies on determining ACR level in food and on the nature
of the ACR because the exposure of humans to ACR can
come from both external sources and the diet.
ACR is reported, at a dose of 7–14 mg/kg, to reduce fertility
rates, increase resorptions of fetuses, reduce litter size in preg-
nant females, and cause the formation of abnormal sperm and
decrease sperm count in males (Sakamoto and Hashimoto
1986; Chapin et al. 1995). ACR-induced histopathological
lesions, such as formation of multinucleated giant cells and
vacuolation, and numerous apoptotic cells were observed in
seminiferous tubules. Serum testosterone level and Leydig cell
viability were also decreased dose-dependently, which resulted
in decreased spermatogenesis (Yang et al. 2005). Although
not active in the in vitro Ames test, ACR does elicit mutagenic
effects in stem cell spermatogonia (Dearfield et al. 1995). ACR
is also reported to induce dominant lethal mutations in sper-
matids (clastogenic or chromosome damaging effects) of mice
and rats and is thus considered to be a mammalian germ cell
mutagen (Shelby et al. 1987; Adler et al. 2002).
The molecular mechanisms of reproductive toxicity could
be the result of alkylation of SH groups in the sperm nucleus
and tail, depletion of GSH, or DNA damage in the testis
(Dearfield et al. 1995). Moreover, the ACR induces chromatin
ISSN 1537-6516 print/ISSN 1537-6516 online © 2009 Informa UK Ltd
DOI: 10.1080/15376510802499048
abstract
This study investigates the simultaneous effect of acrylamide (ACR) and high-fat-intake on the apoptosis in testis
cells, and also the expression and activity of caspase-3. Seventy-two male Kunming mice were divided into two
blocks and fed with a high-fat diet (crude fat 21.5%) or basic diet (crude fat 4.4%), respectively; and animals in
each diet block were exposed to ACR at the dose of 20 mg/kgbw•d or 40 mg/kgbw•d as ACR treated groups or the
normal saline as control. Germ cells prepared from testis were stained with Hoechst dye 33258 and paraffin wax
sections from testis were suffered to a TUNEL process. Expression of caspase-3 on protein level was investigated
using an immunohistochemical analysis assay. The supernatant of unilateral testes were subjected to a Caspase-3
activity kit to determine the activity of Caspase-3 in testis. The concentration of ACR and glycidamide(GA), epox-
ide of ACR, in plasma and testis were detected by LC-ES/MS/MS analysis. Results based on the morphological
changes, percentage of apoptotic cells, and integrated optical density (IOD) of positive amethyst staining which
indicates the apoptotic DNA fragmentation, show that apoptosis was induced by acrylamide only; however, acr-
ylamide-induced apoptosis was weakened by high-fat-intake. The protein expression and activity of Caspase-3
were not induced by ACR or high-fat-intake. Moreover, no significant differences of ACR and GA concentration
were found between the high-fat and basic diet groups after exposure of ACR. Results indicate that high-fat-
intake reverses the effects on apoptosis induced by ACR; and more possibly, apoptosis is induced by a caspase-
3-independent mechanism.
Keywords: Acrylamide, High-fat, Testis, Apoptosis, Caspase-3
abbreviations: ACR, Acrylamide; GA, glycidamide; IOD, integrated optical density
http://www.informapharmascience.com/txm

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220 Xichun Zhang et al.
adducts in germ cells of male mice, which indicates that ACR
can reach sperm cell nuclei (Holland et al. 1999). A cytom-
eter-based dose-response micronucleus assay shows that
very low doses of ACR can damage chromosomes of mouse
spermatids and the linearity of the dose-response suggests
that ACR is DNA-reactive clastogens and represent a health
risk to mouse spermatids (Adler et al. 2000).
Apoptosis, a selective process of physiological cell dele-
tion that regulates the balance between cell proliferation
and cell death, is induced by DNA damage or oxidative
stress. Programmed germ cell death plays an indispensable
role during fetal, neonatal, postnatal, and adult sperma-
togenesis (Mori et al. 1997; Wang et al. 1998; Sinha Hikim
and Swerdloff 1999). During the apoptotic process, caspases
are considered as a central player for the apoptotic process
and cascade of proteolytic cleavage event. The Caspase-
dependent process is associated with two pathways of
apoptosis, a death receptor-dependent and a mitochondria-
dependent pathway (Debatin 2004). Death receptor activa-
tion pathway is mediated with a death-inducing signaling
complex, which is made of a Fas-associated death domain
and a procaspase-8, activating Caspase-8 (Kim et al. 2002).
Caspase-8 directly activates Caspase-3, leading to apoptosis.
The mitochondria-dependent pathway involves cleavage of
proapoptotic proteins by Caspase-8 releasing cytochrome
C in the mitochondria. Released cytochrome C activates
Caspase-3 and Caspase-9, inducing the apoptosis with
morphological, biochemical, and mitochondrial changes
(Li et al. 2003). Caspase-3, in particular, is associated with
the execution of apoptosis and induces the large number of
morphological changes characteristic of cells undergoing
apoptosis (Shi 2002).
Although studies on ACR on reproductive toxicity have
been carried out, the role of apoptosis in this process is still
unclear. Moreover, high-fat-intake, which is common in diet
and always exposes to humans together with ACR through
lots of food such as fried potato, may interacts with ACR on
reproductive toxicity induced by ACR. Epidemiological stud-
ies and laboratory animal model assays suggest that a high
intake of dietary fat promotes mammary carcinogenesis. For
an animal model, both the high-fat corn oil diet and high-fat
mixed-lipid diet promote 7,12-dimethylbenz[a]anthracene-
induced mammary carcinogenesis when compared to a
low-fat corn oil diet (Kuno et al. 2003).
In the present study, we investigate the combining effect
of ACR and high-fat-intake on the apoptosis in testis cells of
Kunming mice. And then we investigate the expression and
activity of caspase-3.
Materials and methods
Animal model and treatment
Kunming mouse is an outbred stock derived from Swiss albino
mice with a high heterogeneity of genes (Sun 2001) and is
widely employed in studies on neuroscience (Yu et al. 1997),
immunology (Feng et al. 2002), genetics (Bu et al. 2002), and
pharmacology (Yang et al. 2002) in Chinese laboratories.
Seventy-two male Kunming mice (Animal Center of Xiamen
University, China), weighing 20–30 g, were housed in a SPF
(specified-pathogens free) animal room maintained at con-
stant temperature (22 ± 2°C) and relative humidity (40–60%)
with a 12 hour light/dark cycle. Animals were assigned to
treatment groups by stratified random sampling (12 mice
per group). The mice were given two kinds of diet with dif-
ferent fat content. The basic diet contains 4.4% fat according
to the requirement of mice; The high-fat diet, in which crude
fat was 21.5%, includes 57.5% basic feed, 10% lard, 20% milk
powder, 10% yolk powder, 2% cholesterol, and 0.5% bile
pigment. Mice in two different diet groups were exposed to
ACR at the dose of 20 mg/kgbw•d or 40 mg/kgbw•d and the
normal saline that was used as control.
Normal saline in which ACR dissolved and also the nor-
mal saline (control) were intraperitoneally injected at a dose
of 10 ml/kgbw (five times per week for 4 weeks).
Blood was collected via cardiac puncture using
heparinized syringes and was immediately processed to
separate the plasma for concentration measurements of
ACR and GA. Testes were removed and immediately frozen
using liquid nitrogen and stored at −80°C for spermatogenic
cell preparation and caspase-3 activity measurements at
a later time; or fixed after perfusion for histochemistry
measurement.
LC-ES/MS/MS analysis of ACR and GA in plasma
and testis
Blood and testis were taken from three mice per acrylamide-
treated group. Analyses of ACR and GA in plasma and tes-
tis were performed using a high-throughput LC-ES/MS/
MS method as previously described (Twaddle et al. 2004).
Briefly, labeled internal standards (13C3-ACR and 13C3-GA)
were added to each thawed plasma sample or testis super-
natant sample (10–100 ml); then samples were purified using
solid phase extraction in 96-well plates and analyzed using
LC-ES/MS/MS in the multiple reaction monitoring mode for
13C-labeled and unlabeled ACR and GA transitions.
Spermatogenic cell preparations and fluorescence
microscopy
Mixed germ cell preparations were obtained by enzymatic
digestion. Briefly, testes were detunicated, digested in
0.5 mg/ml collagenase (Sigma, St. Louis, MO) in Krebs–
Ringer bicarbonate (KRB) media at 32°C for 20 minutes,
then digested with 0.5 mg/ml of trypsin (Sigma) and
1 g/ml DNaseI (USB, Cleveland, OH) in KRB at 32°C for 13
minutes; germ cells were subsequently released by pipetting
for 3 minutes. The germ cell suspension was filtered through
a Nitex mesh and washed three times in 0.5% bovine serum
albumin (BSA; Sigma) in KRB. After the final wash, cells
were counted and resuspended at a concentration of 2.5 ×
106 cells/ml in KRB media; the cells were then centrifuged at
5000 rpm for 5 minutes. Then cells were fixed in 4% parafor-
maldehyde for 5 minutes. The cells were then stained with
Hoechst dye 33258 (8 µg/ml) for 5 minutes, washed twice
with phosphate buffered saline (PBS), and mounted. Nuclei

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Mice testis apoptosis induced by high-fat and ACR 221
were observed using an Axiovert 100 inverted fluorescence
microscope.
Histochemistry assay
Animals were anesthetized with ether, and then perfused
with the normal saline through the vessel at 37°C till the tes-
tes turn white. To keep the tissue shape from changing before
embedding, perfusion was carried out with phosphate buffer
(pH 7.4) containing 4% polyformaldehyde through artery at
4°C for 10 min. Then, testes were taken off on ice, immersed
into 4% polyformaldehyde for 24 hours, dehydrated in
50–100% ethanol, cleared in xylene, and embedded in paraf-
fin wax. The central cross-cut serial sections with a thickness
of 4 m were obtained for every 3 mm of the tissue.
A TUNEL kit (Wako, Osaka, Japan) was used for DNA
fragmentation analysis according to the manufacturer’s
instructions. Briefly, the tissue sections were deparaffinized,
rehydrated and treated with protein digestion buffer for 8
minutes at 37°C, followed by incorporation of biotin-deox-
yuridine triphosphate in the presence of working strength
deoxynucleotidyl transferase (TdT) for 10 minutes at 37°C.
After blocking the endogenous peroxidase (POD) activity by
incubation with 3% hydrogen peroxide (H2O2) in PBS buffer
for 5 minutes at room temperature, the tissue samples were
incubated with POD-conjugated antibody for 10 minutes at
37°C, developed with dimethylaminoazobenzene (DAB) for
5 minutes at room temperature, and finally counterstained
with methyl-green and examined under a MOTIC Biological
Microscope (Micro-optic Industrial Group, Xiamen, PRC).
Cells with amethyst staining, indicative of DNA fragmen-
tation, were considered to indicate apoptosis and the
integrated optical density (IOD) of positive amethyst stain-
ing was analyzed with Images Pro Plus 5.0.1 (Micro-optic
Industrial Group, Xiamen, PRC) to indicate the expression
level of protein.
Tissue sections of sham-injected mice pretreated with
DNase I prior to incubation with TdT were used as a positive
control, while tissue sections pre-treated with TdT substrate
solution without TdT served as a negative control in TUNEL
assay.
Immunohistochemical analysis (IMC) was used to inves-
tigate the apoptotic caspase-dependent pathway. Caspase-3
plays an essential role as an executor in apoptosis (Porter
and Janicke 1999). Therefore, we examined whether ACR
or high-fat-intake activates caspase-3 during the induction
of apoptosis in germ cells. Sections were deparaffinized
in xylene and hydrated in gradient ethanol. Endogenous
peroxidase was blocked with 3% H2O2 in 70% methanol.
Then sections were washed for 10 minutes in PBS (pH7.3),
and non-specific protein-binding sites were blocked with
5% normal goat serum to reduce background staining.
Next, immunolocalization of Caspase-3 were processed
using a monoclonal antibody against Caspase-3 (1:600
dilution; Santa Cruz Biotechnology, Santa Cruz, CA) as the
primary antibodies. Sections were then incubated with the
biotinylated secondary antibody, rabbit anti-mouse IgG,
and then strep avidin-biotin complex (SABC) agent (Boster
biotechnology, Wuhan, PRC) for 20 minutes at 37°C. After
a 5-minute wash, the sections were treated with liquid
diaminobenzidine (Boster biotechnology, Wuhan, PRC)
and counterstained with hematoxylin. The stained sections
were photographed using a MOTIC Biological Microscope
(Micro-optic Industrial Group, Xiamen, PRC). IOD of
positive immunostain was analyzed with Images Pro Plus
5.0.1(Micro-optic Industrial Group, Xiamen, PRC).
Caspase-3 activity
Caspase-3 activity was measured using testis supernatants
with the Caspase-Glo Assay System (Promega, Madison,
WI) according to the manufacturer’s instructions. Briefly,
50 l of the Caspase-Glo buffer was added to 50 l of tis-
sue supernatants in a 96-well plate and incubated at room
temperature for 60 minutes. Luminescence was measured
using a Biotek synergy microplate reader (Biotek, Winooski,
VT). Background activity levels, based on measurements in
homogenization buffer only, were measured and subtracted
from values in the testis tissue supernatants. All values were
normalized to total protein content. Standard curves were
used to determine the linearity of the responses.
Statistical analysis
All data were expressed as mean ± SD. Means of differ-
ent groups were compared with Repeated Measured Two
Factors ANOVA (F-test). Multi-comparison was taken by
LSD or SNK (Q-test).
Results
Concentration of ACR and GA in plasma and testis
Concentration of ACR and GA in the plasma and testis are
presented as mean ± SD of three animals. No significant dif-
ferences of ACR and GA concentration were found between
the high-fat and basic diet groups after exposure of ACR
(Table 1).
Table 1. Acrylamide and glycidamide concentration in the plasma and testis (μg/mg).


NMACR 20 mg/kgbw•d
ACR 40 mg/kgbw•d
HF ACR 20 mg/kgbw•d
ACR 40 mg/kgbw•d
n = number of mice used in experience, NM = normal diet block, HF = high-fat diet block. Acrylamide and glycidamide concentration in the plasma and
testis are presented as mean ± SD of three animals. No significant differences of acrylamide and glycidamide concentration were found between the
high-fat and basic diet groups after exposure of acrylamide.
n
3
3
3
3
ACRGA
Plasma
5.5 ± 2.2
13.4 ± 3.2
6.9 ± 2.3
14.2 ± 2.4
Testis
4.8 ± 3.1
9.2 ± 2.4
6.5 ± 4.0
8.6 ± 1.8
Plasma
4.4 ± 0.4
8.9 ± 0.9
3.5 ± 0.2
7.4 ± 2.0
Testis
2.6 ± 0.1
7.6 ± 1.1
2.3 ± 0.3
7.4 ± 0.5

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222 Xichun Zhang et al.
Apoptosis induced by ACR and high-fat-intake
Apoptosis was first examined by observation of morphologi-
cal changes of cells. Three testes from mice per group were
used to prepare the spermatogenic cells and the cells fixed
in sections were subjected to a fluorescence observation.
The number of cells taking the formation of apoptotic body
were counted. The total cell number was not determined by
dividing cell types such as testicular germ cells and sertoli
cells because it was difficult to determine the difference of
cell types which has no significant differences in nuclear size
(Russell et al. 2002). Thus, the total number of cells reflected
all cell types of seminiferous tubules. Apoptotic change like
formation of apoptotic body was observed under the micro-
scope in ACR-treated mice testis; the percentage of apoptotic
cells in ACR-treated mice testis was significantly higher than
control (ACR-non-exposed group with normal diet). Whereas,
no observed apoptosis and significant changes of percentage
of apoptotic cell were detected out in any mice testis treated
with ACR and high-fat together (Fig. 1A and 1B, Table 2).
Six testes from mice of a group, five sections of a mouse tes-
tes, were carried a TUNEL process. As shown in Fig. 1C and 1D
and Table 2, the apoptosis cells with amethyst staining were
observed in ACR-treated mice testis rather than those treated
with ACR and high-fat together. The IOD of apoptotic cells
(amethyst staining) significantly increased in ACR-treated mice
testis rather than that treated with ACR and high-fat together
(Table 2). Results above indicated that apoptosis was induced
by ACR only rather than ACR and high-fat diet together.
Taken together, data demonstrate that high-fat-intake
weakens the up-regulation of ACR-induced apoptosis in
mouse testes.
Expression and activity of Caspase-3
In order to investigate the apoptotic caspase-dependent
pathway, we used an immunohistochemical analysis assay
to determine the expression of caspase-3 on protein level.
Five sections from every testis of six mice of a group were
carried out for the immunohistochemical analysis. The IOD
was used to indicate the expression level of protein and the
results are shown in Table 3. ACR (20 mg/kgbw•d and 40 mg/
kgbw•d) did not significantly increased the Caspase-3 IOD
compared to control (ACR-non-exposed group with normal
diet). Simultaneous exposure of acrylamide and high-fat-
intake did not significantly chang the Caspase-3 IOD com-
pared to control too. The results above suggest that either
acrylamide or high-fat-intake do not induce the expression
of Caspase-3, as well as that of the combined.
Because the cleavage of procaspase-3 to active
Caspase-3 is essential to the activity of Caspase-3 (Pandey
et al. 2000), we also investigated the activity of Caspase-3 in
testis after detecting expression on protein level. Unilateral
testes of three mice per treated group were homogenized,
and then the supernatants were subjected to a Caspase-3
activity kit. Compared with the control, no group had sig-
nificant change on activity of Caspase-3, which indicated
that either acrylamide or high-fat-intake do not induce the
activity of Caspase-3, as well as that of the combined.
Discussion
The purpose of this present study is mainly to make sure if
high-fat-intake has an altering effect on the reproductive tox-
icity of ACR through the mechanism of apoptosis, as well as
the caspase-dependent pathway during the apoptotic proc-
ess. Results coming from the present study are beneficial
AB
CD
Figure 1. Apoptosis induced by acrylamide or high-fat. Apoptotic body
formation (open arrow) was observed under the microscope in acryla-
mide-treated mice testis (B), but not those treated with acrylamide and
high-fat together (A). And, as shown, the apoptosis cells with amethyst
staining (arrow) were observed in acrylamide-treated mice testis (D), but
not those treated with acrylamide and high-fat together (C).
Table 2. Apoptosis induced by acrylamide and high-fat intake.


NM ACR 0 mg/kgbw•d
ACR 20 mg/kgbw•d
ACR 40 mg/kgbw•d
HF ACR 0 mg/kgbw•d
ACR 20 mg/kgbw•d
ACR 40 mg/kgbw•d
n = number of mice used in experience, NM = normal diet block, HF = high-fat diet block.
a p < 0.05 (compared to NM 0 mg/kgbw•d).
ACR significantly increased the apoptosis which was indicated by the apoptotic cell number of total spermatogenic cells and the IOD of apoptotic
cells (amethyst staining) per animal in acrylamide-treated mice testes. However, no significant change occurred on the apoptotic cell number of total
spermatogenic cells and the IOD of apoptotic cells in mice testes treated with acrylamide and high-fat together compared to control.
Fluorescence microscopy
Apoptotic cell %
TUNEL assay
n
3
3
3
3
3
3
n
6
6
6
6
6
6
Staining cells %
23 ± 2
158 ± 21a
169 ± 23a
26 ± 5
28 ± 4
31 ± 5
IOD of amethyst staining
198.34 ± 56.83
2007.03 ± 1025.61a
4325.37 ± 208.13 a
112.23 ± 98.51
158.41 ± 65.96
162.51 ± 58.27
2 ± 0.5
15 ± 2a
16 ± 3a
3 ± 0.2
4 ± 0.7
3 ± 0.1

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Mice testis apoptosis induced by high-fat and ACR 223
to determine the upper level of ACR in human diets and to
determine the limited standard of ACR in relative foods such
as fried potato chips, also to improve the techniques reduc-
ing ACR in questionably high-ACR foods.
We found the apoptosis induced by ACR in mice testes
(p < 0.05); however, no significant apoptotic change was
observed after the combined exposure of ACR and high-fat-
intake (p > 0.05). As for the caspase-3, no significant pro-
tein expression and activity changes were observed in any
treated group (p > 0.05). Moreover, no significant differences
on ACR or GA concentration were observed between basic
and high-fat diet group after ACR-exposure (p > 0.05). All
the data suggest that high fat intake reverses the apoptosis
induced by ACR; and, more possibly, apoptosis was induced
by a caspase-3-independent mechanism. To our knowledge,
this is the first finding of interaction between ACR and high-
fat-intake. Based on the findings of the present study, we
should reconsider the reproductive toxicity of ACR because
of the high frequency of high-fat diet in daily life.
ACR toxicity appears to increase Leydig cell death and
perturb gene expression levels, contributing to sperm
defects and various abnormal histopathological lesions
including apoptosis in rat testis (Yang et al. 2005). Our study
demonstrated the apoptosis induced by ACR in mice testis.
Acrylamide alone slightly increased apoptosis of bovine lens
epithelial (BEL) cells; however, simultaneous exposure to
ACR and staurosporine for 8 hours produced significantly
less apoptosis than staurosporine alone, and pre-incubation
with ACR followed by staurosporine for 8 and 24 hours mark-
edly reduced apoptosis compared to staurosporine alone
(Arocena 2006). ACR seems therefore to have a dual effect
on BEL cell survival. Moreover, promoting effects of high-
fat diets on chemical-induced mammary tumorigenesis,
which is the result of abnormal apoptosis, was also reported
(Reddy and Sugie 1988). The present study shows a consist-
ent conclusion; simultaneous exposure to ACR and high-fat-
intake produced significantly less apoptosis than ACR alone
in mice testes.
Caspase-3, which is involved in the nuclear fragmenta-
tion at the final step of apoptosis, in particular, is associated
with the execution of apoptosis (Shi 2002). In this study, DNA
fragmentation and apoptotic body were induced in the ACR-
treated group (p < 0.05) and was not induced in the group
treated with ACR and high-fat together (p > 0.05), which was
not consistent with the change of activity of caspase-3, as
well as the capase-3 expression, so apoptosis was induced
more possibly by a capase-3-independent pathway. More
recently, studies show that mechanisms other than caspase-
dependent apoptosis may be involved in the apoptotic
process in the genesis of toxicity of ACR in SH-SY5Y cells
(Tomoyuki and Hideki 2007).
Caspase-3-independent apoptosis, which were induced
by radiofrequency fields in cortical neurons (Joubert et al.
2008), by ciglitazone in renal epithelial cells (Kwon et al. 2008),
by oxidative stress in immortalized ganglion cell line (Li and
Osborne 2008), by phenoxazine derivatives (2-amino-4,4
alpha-dihydro-4alpha-phenoxazine-3-one and 2-aminophe-
noxazine-3-one) in human neuroblastoma cell line NB-1
cells (Shirato et al. 2007), and by porphyromonas gingivalis
in fibroblast (Desta and Graves 2007), were reported more
recently. So, in response to apoptotic stimuli, mitochondria
can release caspase-independent cell death effectors. One
of these effectors is apoptosis inducing factor (AIF), which
could induce nuclear apoptosis in a variety of cell types. The
effect was not inhibited by pharmacological caspase inhibi-
tors such as zVAD, indicating that AIF can trigger nuclear
apoptosis in a caspase-independent manner. AIF binds to
DNA in a sequence-independent manner, which determines
the entry of this complex into the nucleus. It recruits or acti-
vates an endonuclease to facilitate DNA fragmentation and
chromatin condensation (Cregan et al. 2004). Calpain, a
Ca2+-dependent intracellular cysteine protease in cell death,
is also a caspase-independent cell death effector. Calpain
is activated in various necrotic and an apoptotic condition,
while caspase-3 is only activated in apoptosis. Caspases and
calpains share several substrates, including PARP. During
apoptosis, the 116 kDa PARP is degraded by caspase-3 to
distinct 89 and 27 kDa fragments; however, PARP is cleaved
by calpain at alternative sites to generate fragments from 70
to 40 kDa in size during necrosis (McGinnis et al. 1999). So
calpain contributes to cell death and replaces caspase-3 to
execute PARP activation in part (Qiao et al. 2007). In addi-
tion, Caspase-7 might substitute for capase-3 in most cell
types and tissues (Wang 2000), since it is highly homolo-
gous to caspase-3 and has very similar substrate specificity.
The present study failed to determine which other protein,
except for caspase-3, is involved in calming down apoptosis.
The role of proteins discussed above should be investigated
in the future.
To conclude, ACR induces apoptosis in mice testis and
high-fat-intake reverses the effects on ACR-induced apop-
tosis; and the apoptosis is induced more possibly through
a caspase-3-independent pathway which is indicated by no
activity of caspase-3 or lack of capase-3 expression.
Acknowledgment
Declaration of interest: The authors report no conflicts of
interest. The authors alone are responsible for the content
and writing of the paper.
Table 3. Expressions on protein level and activity of Caspase-3.

Protein expression
n
NM ACR 0 mg/kgbw•d63127.83 ± 515.29
ACR 20 mg/kgbw•d62227.60 ± 524.83
ACR 40 mg/kgbw•d61661.40 ± 967.54
HF ACR 0 mg/kgbw•d64619.37 ± 104.48
ACR 20 mg/kgbw•d62809.08 ± 125.36
ACR 40 mg/kgbw•d62498.71 ± 525.74
n = number of mice used in experience, NM = normal diet block, HF =
high-fat diet block. Compared with the control (ACR-non-exposed group
with normal diet), no group had significant change on protein expression
or activity of Caspase-3.
Protein activity
Relative light units
2354.23 ± 125.35
2254.65 ± 235.18
1998.54 ± 238.62
2365.82 ± 135.26
2553.42 ± 155.21
2109.37 ± 366.81
IODn
3
3
3
3
3
3