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Comparative Cytotoxicity and Sperm Motility Using

a Computer-Aided Sperm Analysis System (CASA) for
Isomers of Phthalic Acid, a Common Final Metabolite of
Phthalates
a

Seung Jun Kwack & Byung-Mu Lee

a

Department of Biochemistry and Health Science, College of Natural Sciences, Changwon

National University, Changwon, Gyeongnam, South Korea
b

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Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Suwon, GyeonggiDo, Korea

Published online: 07 Aug 2015.

To cite this article: Seung Jun Kwack & Byung-Mu Lee (2015): Comparative Cytotoxicity and Sperm Motility Using a ComputerAided Sperm Analysis System (CASA) for Isomers of Phthalic Acid, a Common Final Metabolite of Phthalates, Journal of
Toxicology and Environmental Health, Part A: Current Issues, DOI: 10.1080/15287394.2015.1067503
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Journal of Toxicology and Environmental Health, Part A, 00:113, 2015

Copyright Taylor & Francis Group, LLC
ISSN: 1528-7394 print / 1087-2620 online
DOI: 10.1080/15287394.2015.1067503

COMPARATIVE CYTOTOXICITY AND SPERM MOTILITY USING A COMPUTERAIDED SPERM ANALYSIS SYSTEM (CASA) FOR ISOMERS OF PHTHALIC ACID,
A COMMON FINAL METABOLITE OF PHTHALATES
Seung Jun Kwack1, Byung-Mu Lee2

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1
Department of Biochemistry and Health Science, College of Natural Sciences, Changwon
National University, Changwon, Gyeongnam, South Korea
2
Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-Do,
Korea

The general population is exposed to phthalates through consumer products, diet, and
medical devices. Phthalic acid (PA) is a common final metabolite of phthalates, and its isomers include isophthalic acid (IPA), terephthalic acid (TPA), and phthalaldehyde (o-phthalic
acid, OPA). The purpose of this study was to investigate whether PA and PA isomers exert
reproductive toxicity, including altered sperm movement. In vitro cell viability assays were
comparatively performed using Sertoli and liver cell lines. In animal experiments, PA or PA
isomers (10, 100, or 1000 mg/kg) were administered orally to Sprague-Dawley (SD) rats,
and semen samples were analyzed by computer-aided sperm analysis (CASA). PA treatment
produced a significant effect on curvilinear velocity (VCL), straight-line velocity (VSL), mean
velocity or average path velocity (VAP), amplitude of lateral head displacement (ALH), and
frequency of head displacement or beat cross-frequency (BCF), whereas IPA, TPA, and OPA
induced no marked effects. In vitro cell viability assays showed that mouse normal testis cells
(TM4) and human testis cancer cells (NTERA 2 cl. D1) were more sensitive to PA and OPA than
mouse liver normal cells (NCTC clone 1469) and human fetal liver cells (FL 62891). Our study
suggests that PA and PA isomers specifically produced significant in vitro and in vivo reproductive toxicity, particularly sperm toxicity and testis cell cytotoxicity. Of the isomers examined,
PA appeared to be the most toxic and may serve as a surrogate biomarker for reproductive
toxicity following mixed exposure to phthalates.

hexylphthalate (DEHP), dibutyl phthalate

(DBP), benzyl butyl phthalate (BBP), and
diethyl phthalate (DEP)(Albro et al., 1987)
(Figure 1). Isophthalic acid (IPA), terephthalic
acid (TPA), and phthalaldehyde (o-phthalic
acid, OPA) are PA isomers. The mutagenicity of PA has been evaluated by employing
dominant lethal mutation and sperm head
abnormality assays in male Swiss albino mice,
and data obtained indicated that PA is a
germ-cell mutagen (Jha et al., 1998). Although
PA does not induce significant changes in the
incidence of post-implantation loss or in the

Phthalic acid esters (PAE), potential

endocrine-disrupting chemicals (EDC), are
widely used in plastics and other common
consumer products. PAE produce reproductive
and developmental toxicities, among other
adverse health effects (Autian, 1973; Hill
et al., 2003; Koo and Lee, 2005; Chung et al.,
2013; Bhat et al., 2014; Guerra et al., 2014).
Several in vitro and in vivo toxicological studies
demonstrated a wide range of systemic and target organ toxicities (Filho Ido et al., 2013; Yoon
et al., 2014). Phthalic acid (PA) is a final common metabolite of phthalates including diethyl

Address correspondence to Dr. Byung-Mu Lee, Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Seobu-ro
2066, Suwon, Gyeonggi-Do, Korea, 440-746. E-mail: bmlee@skku.edu
1

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S. J. KWACK AND B.-M. LEE

FIGURE 1. Metabolic pathways of phthalates: diethyl hexylphthalate (DEHP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), and
diethyl phthalate (DEP). Phthalates are metabolized to their monomers, and ultimately to phthalic acid (PA, a common metabolite of
phthalates) and glucuronides.

number or sex ratio of fetuses, this compound

decreases the number of ossification centers
of the caudal vertebrae in male fetuses (Ema
et al., 1997). Several studies focused on the
effects of prostaglandins on bone formation, in
particular, the role of prostaglandin synthase
in osteoblastic cells (Kawaguchi et al., 1995;
Pilbeam et al., 1995), suggesting that PA might
potentially affect prostanoid output.
TPA is an industrial chemical intermediate that is used mainly in the manufacturing
of polyester fibers and films (Gibson, 1982;
Cui et al., 2006). Results from repeated-dose
and acute toxicity studies via oral, dermal,
and inhalation routes indicated that TPA produces low toxicity and is nonirritating to the
skin and eyes (Hall et al., 1993; WolkowskiTyl et al., 1982). The primary adverse effect of
high doses of TPA in rats is almost completely
restricted to the urinary tract (Hoshi et al.,
1967; Dai et al., 2005). This chemical is not a
reproductive toxicant, but in a one-generation
reproduction feeding study, postnatal developmental effects were observed in rats (Hoshi
et al., 1968; Li et al., 1999). TPA is not genotoxic and does not markedly alter frequency

of micronucleated polychromatic erythrocytes

(micronuclei) or chromosomal aberrations (Lee
and Lee, 2007).
IPA is mainly used in the synthesis of resins
and is present in packaging fibers and plastics (Illinois Institute of Technology Research
Institute [IITRI], 1998). Because IPA present
in consumer products is bound in a polymer
matrix, the potential for exposure to consumers
is low (IITRI, 1990). In addition, since IPA does
not persist in the environment, the potential
for environmental exposure is also low. IPA and
TPA are structural isomers, with carboxylic acid
groups attached to the benzene ring at the 1,3and 1,4-carbons, respectively. Both IPA and
TPA possess similar physicochemical properties and metabolic pathways and exhibit similar
toxicological properties. IPA exhibits low acute
toxicity by oral, dermal, and inhalation routes
(Staples et al., 1997); it also has negligible
potential for skin irritation and was considered slightly irritating to the eyes. In repeated
dose studies, the target organ was found to
be the kidney. A no-observed-adverse-effect
level (NOAEL) at 250 mg/kg-d IPA for kidney
toxicity (crystalluria, mild hydronephrosis, and

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CYTOTOXICITY AND SPERM MOTILITY OF PHTHALIC ACIDS

pelvic calcification) was reported in rats following repeated oral exposure (Boots et al.,
1976).
OPA is a reagent that forms fluorescent conjugation products with primary amines (Barr
et al., 2003). Oral repeat exposure studies in
rats using doses between 5 and 50 mg/kg body
weight/d showed direct irritation of stomach
lining and lungs with some minor effects on
blood biochemistry. No marked effects were
noted at a dose level of 5 mg/kg body weight/d
(Mayer et al., 1972). This chemical was found
not to produce birth defects, but delayed rat
fetal development in an oral (gavage) study at
a dose of 40 mg/kg body weight/d administered to dams. OPA displayed moderate acute
oral toxicity in rats (lethal dose, 50% [LD50 ]:
121170 mg/kg) and low dermal toxicity in
rabbits (LD50 > 2000 mg/kg, using a 0.55%
solution) (GISAAA, 1967). Table 1 summarizes
the physicochemical properties of PA, TPA, IPA,
and OPA.
Since computer-assisted sperm analysis
(CASA) systems have advanced, analysis of
sperm motility has been increasingly used as an
endpoint in male rodent fertility and toxicology
studies. Several reports described methods of
sample preparation and analysis for assessing sperm motility in lab animals (Klinefelter
et al., 1991; Toth et al., 1991; Chapin et al.,
1992; Slott et al., 1993; Seed et al., 1996;
Rijsselaere et al., 2012; Schleh and Leoni,
2013; Kummer et al., 2013). CASA systems
enable analysis of large numbers of sperm in
a short period, and provide multiple parameters of sperm motion. However, there are few
reports in which the sperm motion parameters generated by CASA systems are optimal for
the evaluation of the potential adverse effects
of chemicals on male fertility. Such parameters
include percent motile sperm, percent progressively motile sperm (progressive motility),
curvilinear velocity (VCL), average path velocity
(VAP), straight-line velocity (VSL), amplitude of
lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), and straightness
(STR). In the present study, epididymal sperm
motility was comparatively determined using a
CASA system and the HamiltonThorne sperm

analyzer (HTM-IVOS) in male rats treated with

PA, IPA, TPA, or OPA. These agents, which
are known to produce male reproductive toxicity by different mechanisms at high exposure
levels, were administered at lower levels to
detect effects, if any, on sperm motility using
the HTM-IVOS.

METHODS AND EXPERIMENTAL

DESIGN
Materials and Reagents
PA, IPA, and TPA were purchased from
Sigma-Aldrich (Munich, Germany). OPA,
phthalic anhydride, was purchased from TCI
(Tokyo, Japan). Bovine serum albumin (BSA)
and medium 199 with Hanks salts, along with
L -glutamine medium, were purchased from
Gibco (Grand Island, NY). Dulbeccos modified
Eagles medium (DMEM), fetal bovine serum
(FBS), trypsinethylenediamine tetraacetic
acid (EDTA), penicillin, streptomycin, and
phosphate-buffered saline (PBS) were also
purchased from Gibco (Grand Island, NY).
Medium 199 was obtained from Invitrogen
(Carlsbad, CA). All other chemicals used in the
study were of analytical grade or higher.

Cell Culture
Mouse normal testis Sertoli cells (TM4),
human testis cancer cells (NTERA 2 cl. D1),
mouse normal liver cells (NCTC clone 1469),
and human fetal liver cells (FL 62891) were purchased from Korea Cellbank (Seoul, Korea), and
cultured in DMEM, DMEM, minimum essential
medium (MEM)-, DMEM, and Isocoves modified Dubeccos medium, respectively (Gibco,
Grand Island, NY). These cells were grown in
plastic flasks in DMEM supplemented with 10%
inactivated FBS and 1% penicillin and streptomycin. The cells were routinely incubated at
37 C in an atmosphere of 5% CO2 . The cell
cultures were incubated with media containing
250 M phthalic acid isomer (PA, IPA, TPA, and
OPA), or an equivalent volume of vehicle, in
the case of the control culture. The cells were

Reference

LD50 (oral, rat)

No-observed-adverse-effect level
(NOAEL; oral, male rats)

Chemical formula (MW)

Physical state
Color
Melting point
Boiling point
Solubility (water)
Use

Structure

GISAAA, 1967

C8 H6 O4 (166.14 g/mol)
Crystalline powder
White
230 C
Not available
1 g/160 ml
Fixative for perfume; industrial
intermediate
7900 mg/kg

Phthalic acid (PA)

Amoco Co., 1975, 1990

C8 H6 O4 (166.14 g/mol)
Crystalline powder
White
425 C
402 C (Sublimes)
15 mg/L
Components of polyester fiber, film, and
fabricated items
>5000 mg/kg
1220 mg/kg (Bladder calculi formation,
hyperplasia of the bladder epithelium)

Terephthalic acid (TPA)

TABLE 1. General Information for Phthalic Acid, Terephthalic Acid, Isophthalic Acid, and Phthalaldehyde

C8 H6 O4 (166.14 g/mol)
Crystalline powder
White
347 C
300 C (Sublimes)
130 mg/L
Components of polyester fiber, film, and
fabricated items
10,400 mg/kg
250 mg/kg/d (Slight increase in the
incidence of crystalluria, mild
hydronephrosis, and pelvic calcification)
Organization for Economic Cooperation
and Development (OECD) SID, 2002

Isophthalic acid (IPA)

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GISAAA, 1967

121170 mg/kg

C8 H6 O2 (134.13 g/mol)
Solid
Light yellow
56 C
266 C (Sublimes)
Low

Phthalaldehyde (OPA)

CYTOTOXICITY AND SPERM MOTILITY OF PHTHALIC ACIDS

subcultured every 2 or 3 d at a subcultivation

ratio of 1:4.

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Cell Viability Assay

The
3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) dye
reduction assay was used to assess cytotoxicity
of the PA isomers. Cells (1 104 cells/ml) were
seeded in 96-well culture plates and maintained in serum-free media for 24 h until they
were adherent, after which they were cultured
in media supplemented with 5% horse serum
(HS) and 10% FBS. Mitochondrial dehydrogenase enzymes in viable cells converted yellow
water-soluble tetrazolium salt (MTT; Sigma,
St. Louis, MO) to dark blue formazan crystals,
which were stored in cellular cytoplasm. The
MTT solution was then removed, and the mesh
was washed twice with 0.5 ml PBS. Dimethyl
sulfoxide (DMSO, 250 l) was added to each
well to dissolve formazan crystals. The plate
was agitated on a shaker for 30 min to enhance
formazan dissolution. A 200-l aliquot was
drawn from each well and transferred into a
96-well tissue culture plate, and spectrophotometric absorbance was measured at 540 nm
using DMSO as blank.
Animals
Six-week-old male Sprague-Dawley rats
(weight: 150170 g) were purchased from
Samtako, Inc. (Seoul, Korea), and acclimatized to lab conditions for a weekday prior to
the experiments. Rats were housed in an animal facility under a 12-h light/dark cycle at
23 2 C with a relative humidity of 50
10%. The rats were fed a standard rat diet
(Samtako, Inc.) and had free access to water.
All animal care was conducted in accordance
with the Sungkyunkwan University Animal Care
Committee guidelines. Rats were divided into
PA, IPA, TPA, and OPA treatment groups at
10, 100, or 1000 mg/kg, and a control group
(corn oil), with 5 rats per group. After 4 wk
of treatment, testes, cauda epididymides, and
spermaducts were dissected from rats under
anesthesia.

Computer-Aided Sperm Analysis (CASA)

Sperm motion analysis and conventional
semen analysis parameters of the male rat
semen sample were measured. Within 1 h
of collection, cauda epididymis was placed
into a petri dish containing 2 ml warmed
medium 199 containing 0.5% BSA for incubation for 2 min at 37 C. A 2-ml aliquot
of fresh semen was loaded into a 10-mmdeep Makler chamber (Vitro Com Inc., Mt
Lakes, NJ), placed on a stage warmer set at
37 C, and was evaluated using a HamiltonThorne integrated visual optic system (HTMIVOS, Hamilton-Thorne Research, Beverly, MA,
version 10.6)
The motion parameters included motility
(percent motile sperm, percent progressively
motile sperm), and velocity (VAP, which is
a mathematically adjusted velocity, VSL, and
VCL). Parameters describing the swimming pattern of spermatozoa based on head movement
included ALH, which corresponds to the mean
width of the head oscillation as the cell swims,
and BCF, which measures the frequency at
which the cell track crosses the cell path in
either direction. STR (equivalent to VSL/VAP
100) and linearity (equivalent to VSL/VCL
100) are also used to describe the swimming
pattern of the sperm. VAP, VSL, STR, and LIN
are indicators of sperm progression, whereas
VCL, ALH, and BCF are indicators of sperm
vigor.
Statistics
All results are expressed as mean standard deviation. All data were analyzed by oneway analysis of variance (ANOVA) using SPSS
software (SPSS, Cary, NC) followed by Tukeys
post hoc comparisons. Values of p < .05 were
considered significant.

RESULTS
In the in vitro study, cytotoxicity induced by
PA, IPA, TPA, and OPA in TM4, NTERA 2 cl.
D1, NCTC clone 1469, and FL 62891 cell lines
were comparatively evaluated by MTT assay

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S. J. KWACK AND B.-M. LEE

FIGURE 2. Cell viability (%) in (A) mouse normal Sertoli cells (TM4), (B) human testis cancer cells (NTERA 2 cl. D1), (C) normal mouse
liver cells (NCTC clone 1469), and (D) human fetal liver cells (FL 62891), after treatment with phthalic acid (PA), isophthalic acid (IPA),
terephthalic acid (TPA), and o-phthalic acid (OPA), at various concentrations.

(Figure 2). Cell viability was reduced in all cases

in a concentration-dependent manner (101 ,
102 , 103 M). Figure 2 shows the survival rates
of cell cultures in the 4 types of cells after treatment for 24 h. The average percet TM4 cell
viability values after 101 M exposure to PA,
OPA, TPA, and IPA were 60.27 1.2, 75.14
2.2, 69.73 1.4, and 77.41 1.6, respectively. IPA was observed to be the least toxic,
whereas PA was the most toxic. OPA was less
toxic than TPA, but more toxic than IPA.
The average percent NTERA 2 cl. D1 cell
viability values after 101 M exposure to PA,
OPA, TPA, and IPA were 61.11 1.5, 71.85
1.2, 68.15 1.1, and 73.85 1.9, respectively. IPA was observed to be the least toxic,
whereas PA was the most toxic. OPA was less
toxic than TPA, but more toxic than IPA.
The average percent NCTC clone 1469 cell
viability values after 101 M exposure to
PA, OPA, TPA, and IPA were 77.89 1.5,
77.78 1.9, 81.15 1.4, and 77.85
1.5, respectively. Cell viability did not significantly decrease. In addition, the average percent FL 62891 cell viability was not significant.

As shown in Figure 1, cell viability of PAtreated TM4 and NTERA 2 cl. cells significantly
decreased following treatment at the highest
concentrations. Toxicity increased with prolonged exposure with PA toxicity to TM4 and
NTERA 2 cl. D1 cells, being approximately 20%
greater than that of IPA, TPA, and OPA at the
highest concentration (101 M). The toxicity of
PA was more apparent in TM4 and NTERA 2 cl.
D1 cell lines than in liver cell lines, indicating
specificity for reproductive cells.
After 4 wk of treatment with PA or PA isomers, testis weight and percent testis to body
weight values of Sprague-Dawley rats did not
significantly change in PA, IPA, TPA, and OPA
treatment groups. The weight of epididymides
and percet epididymides to body weight values were not affected (Table 2). However, only
the group treated with PA showed a significantly reduced percent body weight of testis
(1.0153) at the highest dose (1000 mg/kg) compared to control (1.4153). Therefore, sperm
analysis was comparatively carried out on the
1000 mg/kg treatment groups. As shown in
Table 3, PA was observed to significantly lower

CYTOTOXICITY AND SPERM MOTILITY OF PHTHALIC ACIDS

TABLE 2. Quantitative Parameters of Epididymal Sperm Analyses in Sprague-Dawley Rats After 4 wk of Daily Oral Treatment at
1000 mg/kg
Group

Control

PA

OPA

TPA

IPA

Testis weight (g)

Percent body weight of testis
Epididymides weight (g)
Percent body weight of epididymides
Sperm concentration (millions/ml)

3.788 0.53
1.4153
1.04 0.13
0.5653
7.66 2.53

3.588 0.93
1.0153
0.99 0.07
0.5087
7.53 1.96

3.688 0.43
1.3453
1.05 0.05
0.5295
8.49 3.05

3.775 0.83
1.2153
1.05 0.07
0.5275
7.72 1.94

3.675 0.53
1.42553
1.01 0.10
0.5512
8.28 1.86

Note. PA, phthalic acid; IPA, isophthalic acid; OPA, phthalaldehyde; TPA, terephthalic acid. Significance: p < .05.

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TABLE 3. Effect of PA, IPA, TPA, and OPA on Sperm Motion Parameters in Sprague-Dawley Rats After 4 wk of Daily Oral Treatment at
1000 mg/kg
Sperm motion
parameters
VAP
VSL
VCL
ALH
BCF
STR
LIN

m/s
m/s
m/s
m
Hz
%
%

Control
(mean SD)

PA
(mean SD)

IPA
(mean SD)

TPA
(mean SD)

OPA
(mean SD)

96.8 7.35
87.9 14.7
215.0 22.8
26.7 2.0
35.2 3.7
90.8 2.6
40.9 2.5

61.2 6.0
31.3 3.0
125.1 16.5
18.6 0.9
26.2 0.9
51.1 2.4
25.0 1.4

71.44 5.2
57.8 9.7
140.0 40.3
22.4 1.8
34.6 4.1
80.9 2.0
41.3 2.1

73.5 4.3
66.6 1.02
175.8 31.6
23.8 1.0
32.6 2.9
90.6 1.2
37.9 2.0

79.9 5.8
57.2 5.7
166.7 13.6
24.5 1.9
30.4 3.5
71.6 2.3
34.3 1.2

Note. ALH, amplitude of lateral head displacement; BCF, beat cross frequency; IPA, isophthalic acid; LIN, linearity (LIN = VSL/VCL
100); OPA, phthalaldehyde; PA, phthalic acid; STR, Straightness (STR = VSL/VAP 100); TPA, terephthalic acid; VAP, velocity average
path; VCL, velocity curved line; VSL, velocity straight line; SD, standard deviation. Significance: p < .05.

VCL, VSL, VAP, LIN, and STR, whereas no significant effects were detected in the parameters
of ALH and frequency of head displacement
(or BCF). No significant alterations in VCL,
VAP, and VSL were observed in epididymal
sperm at 1000 mg/kg/d IPA, TPA, or OPA
treatment. In addition, STR and LIN remained
unchanged. Administration of PA at the highest dose (1000 mg/kg/d) for 4 wk reduced
total and progressive motility of sperm from the
cauda epidiymis; this decrease, however, was
not significant.
Sperm mobility (%) fell in a dosedependent manner in all treatment groups, but
a significant decrease was observed only in
the PA treatment group at the highest dose
(1000 mg/kg) (Figure 3A). Progressive motility
of the sperm (%) also significantly fell only
at the highest concentrations (1000 mg/kg) of
each treatment (Figure 3B). In addition, the
percent of sperm with rapid motion in the
1000-mg/kg/d PA dose group was only 80%
of control, while static sperm (%) significantly
increased in the PA treatment group (100,
1000 mg/kg) (Figures 3C and 3D).

DISCUSSION
Of
polyvinyl
chloride
plasticizers,
phthalates and phthalate monoesters, primarily monoethylhexyl phthalate (MEHP) and
monobutyl phthalate (MBP), are the most
commonly used phthalate esters in medical
devices, food containers, and cosmetic products and may adversely affect reproductive
or developmental functions in animals (Albro,
1987; Boekelheide, 1993; Parks et al., 2000;
Gray et al., 2000; Ema et al., 2003; Bhat et al.,
2014). The Sertoli cell is the primary testicular
target of phthalate esters, and decreases in
testicular weight and spermatid numbers in
neonatal rats were observed following administration of 2000 mg/kg DEHP for 17 wk (Dostal
et al., 1988). Although Sertoli cells are the
direct targets of MEHP, the primary consequence of exposure to rodents is a marked
increase in germ-cell apoptosis (Richburg and
Boekelheide, 1996).
Phthalates undergo rapid metabolism, and
share PA as a common metabolite, in addition to forming their own specific metabolites

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S. J. KWACK AND B.-M. LEE

FIGURE 3. Sperm motility (A), progressive motility of sperm (B), static (C), and rapid (D) movement of sperm from the epididymides of
rats treated with phthalic acid (PA), isophthalic acid (IPA), terephthalic acid (TPA), and o-phthalic acid (OPA) at 10, 100, or 1000 mg/kg/d
for 4 wk, respectively.

(Albro et al., 1987) (Figure 3). Albro and

Thomas (1973) reported that PA was one of
the metabolic products excreted in urine when
DEHP was administered orally to rats. PA, when
administered orally to rats, is neither appreciably metabolized nor retained in the organs or
tissues (Williams and Blanchifield, 1974). Lim
et al. (2007) found that the terminal elimination half-life (t1/2 ) of PA was 57 h. Treatment of
adult male Swiss albino mice with PA resulted
in induction of dominant lethal mutations and
induced dose-dependent increases in abnormal sperm during meiotic and postmeiotic
stages of spermatogenesis. The results obtained
indicate that PA is a germ-cell mutagen (Jha
et al., 1998). PA isomers are suspected to be
potent androgen receptor antagonists and may
produce abnormalities in the male reproductive system (Japan Environment Agency [JEA],
1998; Sharpe, 1998). Pavan et al. (2001)
noted that molecular mechanisms underlying
PA and steroid-hormone responses in the WISH
cell were associated with estrogen-receptor
binding.
There were no adverse effects on fetal
development in mice administered a single
injection of OPA during pregnancy. There were
no evidence of OPA-mediated carcinogenicity

in chronic feeding studies in rats and mice, and

no mutations were detected in bacterial tests
(BIBRA Working Group, 1989).
Our in vitro study showed that direct
cytotoxicity induced by PA isomers was not
likely to be the cause of adverse effects
observed in testes of affected animals. Cell viability was assessed in TM4, NTERA 2 cl. D1,
NCTC clone 1469, and FL 62891 cells using
the MTT assay, but no significant cytotoxicy
was observed in NCTC clone 1469 and FL
62891 cells. However, cytotoxicity was noted
at various concentrations of PA isomers in
TM4 and NTERA 2 cl. D1 cells. The testicular
cells appeared to be more sensitive to toxicants
than are liver cells. In addition, PA-induced
cytotoxicity was more potent than IPA, TPA,
and OPA.
The animal experiment in this study was
used to explore relationships between CASA
parameters and PA isomers. Although results
were not statistically significant except at
a PA concentration of 1000 mg/kg/d, an
overall pattern of decline was observed in
CASA parameters VSL, VCL, and LIN for
phthalic isomers PA, IPA, TPA, and OPA. The
absence of consistent, statistically significant
dose-response relationships may reflect a lack

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CYTOTOXICITY AND SPERM MOTILITY OF PHTHALIC ACIDS

of power due to the relatively small number of

animals.
Several studies demonstrated a relationship between CASA parameters and chemical
exposure. Epichlorohydrin, after 4-h inhalation exposure, transiently reduced path velocity
despite no significant change in percent motile
sperm (Slott et al., 1990). Alpha-chlorohydrin
administered to male hamsters for 4 d resulted
in a significant dose-dependent decline in VCL,
VAP, and VCL, despite no change in percent
motile sperm. In addition, alpha-chlorohydrin
exposure was associated with a nonlinear
impairment for in vitro fertilizing ability, which
exhibited a threshold-like response. Jelks et al.
(2001) examined alpha-chlorohydrin (ACH) at
different concentrations (5, 10, 25, 50, and
75 mg/kg, po) in rats, and effects on sperm
ATP levels, sperm motility, and ability of sperm
to bind and penetrate rat oocytes. Computer
analysis of sperm motility indicated that VSL
was the most sensitive parameter to ACH treatment; VSL significantly decreased in rat sperm
3 h after exposure (25 mg/kg). Data suggest that
CASA parameters may serve as a more sensitive marker of reproductive toxicity than semen
parameters (Perreault and Cancel, 2001). One
mechanism by which sperm motion or testes
may be impaired includes oxidative stress,
the production of reactive oxygen species
(ROS), and subsequent lipid peroxidation of
sperm plasma membrane although the probable mechanisms may depend on type of
chemicals (Storey, 1997; Armstrong et al.,
1999; Martinez et al., 2014; Almiov et al.,
2014).
Ono et al. (1999) examined the association between toluene levels and epididymal
sperm counts, sperm motility, and sperm quality. Data demonstrated that high concentrations of toluene might directly target sperm
in the epididymis and disrupt sperm maturation. Selevan et al. (2000) also examined the
association between air pollution levels and
VSL, VCL, and LIN, and found that medium
levels of air pollution adversely affected VCL,
but improved LIN. Adamkovicova et al. (2012)
reported that cadmium and diazinon administration to rats led to a significant increase

in ALH and significant fall in BCF by using

a CASA system. Although CASA parameters
may prove to be sensitive biomarkers of reproductive toxicity in vivo, these are difficult to
compare across studies because of the use of
different CASA instruments and settings (Davis
et al., 1992; Castellini et al., 2011; Mortimer
and Mortimer, 2013). Despite this limitation, in
vivo studies demonstrated that CASA parameters may be used to predict fertility (Youn
et al., 2011; Frour et al., 2012; Broekhuijse
et al., 2012) and pregnancy (Macleod and
Irvine, 1995; Larsen et al., 2000; Isobe, 2012).
Further, epidemiologic studies have used CASA
parameters as a marker of altered semen quality (Mukhopadhyay et al., 2010; Vested et al.,
2011).
The concentration of PA in biological samples may be useful as a sensitive biomarker of
reproductive toxicity, or as an indirect indicator to estimate prevalence of total exposure to
phthalates and marker of altered semen quality and mobility. In vitro cell viability assays
showed that TM4 and NTERA 2 cl. D1 cells
were more sensitive to PA and OPA than NCTC
clone 1469 and FL62891 cells. Data suggest
that PA and PA isomers demonstrated specific
and significant in vitro and in vivo reproductive
toxicity, specifically, sperm toxicity and testis
cell cytotoxicity. Of the PA isomers, PA seems
to be the most toxic and may serve as a surrogate biomarker for reproductive toxicity upon
mixed exposure to phthalates.
FUNDING
This research was supported by a grant
(14172MFDS975) from the Ministry of Food &
Drug Safety (MFDS) in 2014.
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