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Comparative Cytotoxicity and Sperm Motility Usinga Computer-Aided Sperm Analysis System (CASA) forIsomers of Phthalic Acid, a Common Final Metabolite ofPhthalatesSeung Jun Kwacka & Byung-Mu Leeb

a Department of Biochemistry and Health Science, College of Natural Sciences, ChangwonNational University, Changwon, Gyeongnam, South Koreab Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-Do, KoreaPublished online: 07 Aug 2015.

To cite this article: Seung Jun Kwack & Byung-Mu Lee (2015): Comparative Cytotoxicity and Sperm Motility Using a Computer-Aided Sperm Analysis System (CASA) for Isomers of Phthalic Acid, a Common Final Metabolite of Phthalates, Journal ofToxicology 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, 00:1–13, 2015Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 onlineDOI: 10.1080/15287394.2015.1067503

COMPARATIVE CYTOTOXICITY AND SPERM MOTILITY USING A COMPUTER-AIDED SPERM ANALYSIS SYSTEM (CASA) FOR ISOMERS OF PHTHALIC ACID,A COMMON FINAL METABOLITE OF PHTHALATES

Seung Jun Kwack1, Byung-Mu Lee2

1Department of Biochemistry and Health Science, College of Natural Sciences, ChangwonNational University, Changwon, Gyeongnam, South Korea2Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-Do,Korea

The general population is exposed to phthalates through consumer products, diet, andmedical devices. Phthalic acid (PA) is a common final metabolite of phthalates, and its iso-mers include isophthalic acid (IPA), terephthalic acid (TPA), and phthalaldehyde (o-phthalicacid, OPA). The purpose of this study was to investigate whether PA and PA isomers exertreproductive toxicity, including altered sperm movement. In vitro cell viability assays werecomparatively performed using Sertoli and liver cell lines. In animal experiments, PA or PAisomers (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 treatmentproduced a significant effect on curvilinear velocity (VCL), straight-line velocity (VSL), meanvelocity or average path velocity (VAP), amplitude of lateral head displacement (ALH), andfrequency of head displacement or beat cross-frequency (BCF), whereas IPA, TPA, and OPAinduced 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 thanmouse liver normal cells (NCTC clone 1469) and human fetal liver cells (FL 62891). Our studysuggests that PA and PA isomers specifically produced significant in vitro and in vivo reproduc-tive 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 reproductivetoxicity following mixed exposure to phthalates.

Phthalic acid esters (PAE), potentialendocrine-disrupting chemicals (EDC), arewidely used in plastics and other commonconsumer products. PAE produce reproductiveand developmental toxicities, among otheradverse health effects (Autian, 1973; Hillet 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 studiesdemonstrated a wide range of systemic and tar-get organ toxicities (Filho Ido et al., 2013; Yoonet al., 2014). Phthalic acid (PA) is a final com-mon metabolite of phthalates including diethyl

Address correspondence to Dr. Byung-Mu Lee, Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Seobu-ro2066, Suwon, Gyeonggi-Do, Korea, 440-746. E-mail: bmlee@skku.edu

hexylphthalate (DEHP), dibutyl phthalate(DBP), benzyl butyl phthalate (BBP), anddiethyl phthalate (DEP)(Albro et al., 1987)(Figure 1). Isophthalic acid (IPA), terephthalicacid (TPA), and phthalaldehyde (o-phthalicacid, OPA) are PA isomers. The mutagenic-ity of PA has been evaluated by employingdominant lethal mutation and sperm headabnormality assays in male Swiss albino mice,and data obtained indicated that PA is agerm-cell mutagen (Jha et al., 1998). AlthoughPA does not induce significant changes in theincidence of post-implantation loss or in the

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

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

number or sex ratio of fetuses, this compounddecreases the number of ossification centersof the caudal vertebrae in male fetuses (Emaet al., 1997). Several studies focused on theeffects of prostaglandins on bone formation, inparticular, the role of prostaglandin synthasein osteoblastic cells (Kawaguchi et al., 1995;Pilbeam et al., 1995), suggesting that PA mightpotentially affect prostanoid output.

TPA is an industrial chemical intermedi-ate that is used mainly in the manufacturingof polyester fibers and films (Gibson, 1982;Cui et al., 2006). Results from repeated-doseand acute toxicity studies via oral, dermal,and inhalation routes indicated that TPA pro-duces low toxicity and is nonirritating to theskin and eyes (Hall et al., 1993; Wolkowski-Tyl et al., 1982). The primary adverse effect ofhigh doses of TPA in rats is almost completelyrestricted to the urinary tract (Hoshi et al.,1967; Dai et al., 2005). This chemical is not areproductive toxicant, but in a one-generationreproduction feeding study, postnatal develop-mental effects were observed in rats (Hoshiet al., 1968; Li et al., 1999). TPA is not geno-toxic and does not markedly alter frequency

of micronucleated polychromatic erythrocytes(micronuclei) or chromosomal aberrations (Leeand Lee, 2007).

IPA is mainly used in the synthesis of resinsand is present in packaging fibers and plas-tics (Illinois Institute of Technology ResearchInstitute [IITRI], 1998). Because IPA presentin consumer products is bound in a polymermatrix, the potential for exposure to consumersis low (IITRI, 1990). In addition, since IPA doesnot persist in the environment, the potentialfor environmental exposure is also low. IPA andTPA are structural isomers, with carboxylic acidgroups attached to the benzene ring at the 1,3-and 1,4-carbons, respectively. Both IPA andTPA possess similar physicochemical proper-ties and metabolic pathways and exhibit similartoxicological properties. IPA exhibits low acutetoxicity by oral, dermal, and inhalation routes(Staples et al., 1997); it also has negligiblepotential for skin irritation and was consid-ered slightly irritating to the eyes. In repeateddose studies, the target organ was found tobe the kidney. A no-observed-adverse-effectlevel (NOAEL) at 250 mg/kg-d IPA for kidneytoxicity (crystalluria, mild hydronephrosis, and

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CYTOTOXICITY AND SPERM MOTILITY OF PHTHALIC ACIDS 3

pelvic calcification) was reported in rats fol-lowing repeated oral exposure (Boots et al.,1976).

OPA is a reagent that forms fluorescent con-jugation products with primary amines (Barret al., 2003). Oral repeat exposure studies inrats using doses between 5 and 50 mg/kg bodyweight/d showed direct irritation of stomachlining and lungs with some minor effects onblood biochemistry. No marked effects werenoted at a dose level of 5 mg/kg body weight/d(Mayer et al., 1972). This chemical was foundnot to produce birth defects, but delayed ratfetal development in an oral (gavage) study ata dose of 40 mg/kg body weight/d adminis-tered to dams. OPA displayed moderate acuteoral toxicity in rats (lethal dose, 50% [LD50]:121–170 mg/kg) and low dermal toxicity inrabbits (LD50 > 2000 mg/kg, using a 0.55%solution) (GISAAA, 1967). Table 1 summarizesthe physicochemical properties of PA, TPA, IPA,and OPA.

Since computer-assisted sperm analysis(CASA) systems have advanced, analysis ofsperm motility has been increasingly used as anendpoint in male rodent fertility and toxicologystudies. Several reports described methods ofsample preparation and analysis for assess-ing sperm motility in lab animals (Klinefelteret 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 systemsenable analysis of large numbers of sperm ina short period, and provide multiple parame-ters of sperm motion. However, there are fewreports in which the sperm motion parame-ters generated by CASA systems are optimal forthe evaluation of the potential adverse effectsof chemicals on male fertility. Such parametersinclude percent motile sperm, percent pro-gressively motile sperm (progressive motility),curvilinear velocity (VCL), average path velocity(VAP), straight-line velocity (VSL), amplitude oflateral head displacement (ALH), beat cross fre-quency (BCF), linearity (LIN), and straightness(STR). In the present study, epididymal spermmotility was comparatively determined using aCASA system and the Hamilton–Thorne sperm

analyzer (HTM-IVOS) in male rats treated withPA, IPA, TPA, or OPA. These agents, whichare known to produce male reproductive toxi-city by different mechanisms at high exposurelevels, were administered at lower levels todetect effects, if any, on sperm motility usingthe HTM-IVOS.

METHODS AND EXPERIMENTALDESIGN

Materials and ReagentsPA, 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 withL-glutamine medium, were purchased fromGibco (Grand Island, NY). Dulbecco’s modifiedEagle’s medium (DMEM), fetal bovine serum(FBS), trypsin–ethylenediamine tetraaceticacid (EDTA), penicillin, streptomycin, andphosphate-buffered saline (PBS) were alsopurchased from Gibco (Grand Island, NY).Medium 199 was obtained from Invitrogen(Carlsbad, CA). All other chemicals used in thestudy were of analytical grade or higher.

Cell CultureMouse 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 pur-chased from Korea Cellbank (Seoul, Korea), andcultured in DMEM, DMEM, minimum essentialmedium (MEM)-α, DMEM, and Isocove’s mod-ified Dubecco’s medium, respectively (Gibco,Grand Island, NY). These cells were grown inplastic flasks in DMEM supplemented with 10%inactivated FBS and 1% penicillin and strepto-mycin. The cells were routinely incubated at37◦C in an atmosphere of 5% CO2. The cellcultures were incubated with media containing250 μM phthalic acid isomer (PA, IPA, TPA, andOPA), or an equivalent volume of vehicle, inthe case of the control culture. The cells were

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TABL

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Gen

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CYTOTOXICITY AND SPERM MOTILITY OF PHTHALIC ACIDS 5

subcultured every 2 or 3 d at a subcultivationratio of 1:4.

Cell Viability AssayThe 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) dyereduction assay was used to assess cytotoxicityof the PA isomers. Cells (1 × 104 cells/ml) wereseeded in 96-well culture plates and main-tained in serum-free media for 24 h until theywere adherent, after which they were culturedin media supplemented with 5% horse serum(HS) and 10% FBS. Mitochondrial dehydroge-nase enzymes in viable cells converted yellowwater-soluble tetrazolium salt (MTT; Sigma,St. Louis, MO) to dark blue formazan crystals,which were stored in cellular cytoplasm. TheMTT solution was then removed, and the meshwas washed twice with 0.5 ml PBS. Dimethylsulfoxide (DMSO, 250 μl) was added to eachwell to dissolve formazan crystals. The platewas agitated on a shaker for 30 min to enhanceformazan dissolution. A 200-μl aliquot wasdrawn from each well and transferred into a96-well tissue culture plate, and spectrophoto-metric absorbance was measured at 540 nmusing DMSO as blank.

AnimalsSix-week-old male Sprague-Dawley rats

(weight: 150–170 g) were purchased fromSamtako, Inc. (Seoul, Korea), and acclima-tized to lab conditions for a weekday prior tothe experiments. Rats were housed in an ani-mal facility under a 12-h light/dark cycle at23 ± 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 accordancewith the Sungkyunkwan University Animal CareCommittee guidelines. Rats were divided intoPA, IPA, TPA, and OPA treatment groups at10, 100, or 1000 mg/kg, and a control group(corn oil), with 5 rats per group. After 4 wkof treatment, testes, cauda epididymides, andspermaducts were dissected from rats underanesthesia.

Computer-Aided Sperm Analysis (CASA)Sperm motion analysis and conventional

semen analysis parameters of the male ratsemen sample were measured. Within 1 hof collection, cauda epididymis was placedinto a petri dish containing 2 ml warmedmedium 199 containing 0.5% BSA for incu-bation for 2 min at 37◦C. A 2-ml aliquotof fresh semen was loaded into a 10-mm-deep Makler chamber (Vitro Com Inc., MtLakes, NJ), placed on a stage warmer set at37◦C, and was evaluated using a Hamilton-Thorne integrated visual optic system (HTM-IVOS, Hamilton-Thorne Research, Beverly, MA,version 10.6)

The motion parameters included motility(percent motile sperm, percent progressivelymotile sperm), and velocity (VAP, which isa mathematically adjusted velocity, VSL, andVCL). Parameters describing the swimming pat-tern of spermatozoa based on head movementincluded ALH, which corresponds to the meanwidth of the head oscillation as the cell swims,and BCF, which measures the frequency atwhich the cell track crosses the cell path ineither direction. STR (equivalent to VSL/VAP× 100) and linearity (equivalent to VSL/VCL ×100) are also used to describe the swimmingpattern of the sperm. VAP, VSL, STR, and LINare indicators of sperm progression, whereasVCL, ALH, and BCF are indicators of spermvigor.

StatisticsAll results are expressed as mean ± stan-

dard deviation. All data were analyzed by one-way analysis of variance (ANOVA) using SPSSsoftware (SPSS, Cary, NC) followed by Tukey’spost hoc comparisons. Values of p < .05 wereconsidered significant.

RESULTS

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

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FIGURE 2. Cell viability (%) in (A) mouse normal Sertoli cells (TM4), (B) human testis cancer cells (NTERA 2 cl. D1), (C) normal mouseliver 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 casesin a concentration-dependent manner (10−1,10−2, 10−3 M). Figure 2 shows the survival ratesof cell cultures in the 4 types of cells after treat-ment for 24 h. The average percet TM4 cellviability values after 10−1 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, respec-tively. IPA was observed to be the least toxic,whereas PA was the most toxic. OPA was lesstoxic than TPA, but more toxic than IPA.

The average percent NTERA 2 cl. D1 cellviability values after 10−1 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, respec-tively. IPA was observed to be the least toxic,whereas PA was the most toxic. OPA was lesstoxic than TPA, but more toxic than IPA.

The average percent NCTC clone 1469 cellviability values after 10−1 M exposure toPA, 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 signifi-cantly decrease. In addition, the average per-cent FL 62891 cell viability was not significant.

As shown in Figure 1, cell viability of PA-treated TM4 and NTERA 2 cl. cells significantlydecreased following treatment at the highestconcentrations. Toxicity increased with pro-longed exposure with PA toxicity to TM4 andNTERA 2 cl. D1 cells, being approximately 20%greater than that of IPA, TPA, and OPA at thehighest concentration (10−1 M). The toxicity ofPA was more apparent in TM4 and NTERA 2 cl.D1 cell lines than in liver cell lines, indicatingspecificity for reproductive cells.

After 4 wk of treatment with PA or PA iso-mers, testis weight and percent testis to bodyweight values of Sprague-Dawley rats did notsignificantly change in PA, IPA, TPA, and OPAtreatment groups. The weight of epididymidesand percet epididymides to body weight val-ues were not affected (Table 2). However, onlythe group treated with PA showed a signifi-cantly reduced percent body weight of testis(1.0153) at the highest dose (1000 mg/kg) com-pared to control (1.4153). Therefore, spermanalysis was comparatively carried out on the1000 mg/kg treatment groups. As shown inTable 3, PA was observed to significantly lower

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CYTOTOXICITY AND SPERM MOTILITY OF PHTHALIC ACIDS 7

TABLE 2. Quantitative Parameters of Epididymal Sperm Analyses in Sprague-Dawley Rats After 4 wk of Daily Oral Treatment at1000 mg/kg

Group Control PA OPA TPA IPA

Testis weight (g) 3.788 ± 0.53 3.588 ± 0.93 3.688 ± 0.43 3.775 ± 0.83 3.675 ± 0.53Percent body weight of testis 1.4153 1.0153∗ 1.3453 1.2153 1.42553Epididymides weight (g) 1.04 ± 0.13 0.99 ± 0.07 1.05 ± 0.05 1.05 ± 0.07 1.01 ± 0.10Percent body weight of epididymides 0.5653 0.5087 0.5295 0.5275 0.5512Sperm concentration (millions/ml) 7.66 ± 2.53 7.53 ± 1.96 8.49 ± 3.05 7.72 ± 1.94 8.28 ± 1.86

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

TABLE 3. Effect of PA, IPA, TPA, and OPA on Sperm Motion Parameters in Sprague-Dawley Rats After 4 wk of Daily Oral Treatment at1000 mg/kg

Sperm motionparameters

Control(mean ± SD)

PA(mean ± SD)

IPA(mean ± SD)

TPA(mean ± SD)

OPA(mean ± SD)

VAP μm/s 96.8 ± 7.35 61.2 ± 6.0∗ 71.44 ± 5.2 73.5 ± 4.3 79.9 ± 5.8VSL μm/s 87.9 ± 14.7 31.3 ± 3.0∗ 57.8 ± 9.7 66.6 ± 1.02 57.2 ± 5.7VCL μm/s 215.0 ± 22.8 125.1 ± 16.5∗ 140.0 ± 40.3 175.8 ± 31.6 166.7 ± 13.6ALH μm 26.7 ± 2.0 18.6 ± 0.9 22.4 ± 1.8 23.8 ± 1.0 24.5 ± 1.9BCF Hz 35.2 ± 3.7 26.2 ± 0.9 34.6 ± 4.1 32.6 ± 2.9 30.4 ± 3.5STR % 90.8 ± 2.6 51.1 ± 2.4∗ 80.9 ± 2.0 90.6 ± 1.2 71.6 ± 2.3LIN % 40.9 ± 2.5 25.0 ± 1.4∗ 41.3 ± 2.1 37.9 ± 2.0 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 averagepath; VCL, velocity curved line; VSL, velocity straight line; SD, standard deviation. Significance: ∗p < .05.

VCL, VSL, VAP, LIN, and STR, whereas no sig-nificant effects were detected in the parametersof ALH and frequency of head displacement(or BCF). No significant alterations in VCL,VAP, and VSL were observed in epididymalsperm at 1000 mg/kg/d IPA, TPA, or OPAtreatment. In addition, STR and LIN remainedunchanged. Administration of PA at the high-est dose (1000 mg/kg/d) for 4 wk reducedtotal and progressive motility of sperm from thecauda epidiymis; this decrease, however, wasnot significant.

Sperm mobility (%) fell in a dose-dependent manner in all treatment groups, buta significant decrease was observed only inthe PA treatment group at the highest dose(1000 mg/kg) (Figure 3A). Progressive motilityof the sperm (%) also significantly fell onlyat the highest concentrations (1000 mg/kg) ofeach treatment (Figure 3B). In addition, thepercent of sperm with rapid motion in the1000-mg/kg/d PA dose group was only 80%of control, while static sperm (%) significantlyincreased in the PA treatment group (100,1000 mg/kg) (Figures 3C and 3D).

DISCUSSION

Of polyvinyl chloride plasticizers,phthalates and phthalate monoesters, pri-marily monoethylhexyl phthalate (MEHP) andmonobutyl phthalate (MBP), are the mostcommonly used phthalate esters in medicaldevices, food containers, and cosmetic prod-ucts and may adversely affect reproductiveor 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 testiculartarget of phthalate esters, and decreases intesticular weight and spermatid numbers inneonatal rats were observed following adminis-tration of 2000 mg/kg DEHP for 17 wk (Dostalet al., 1988). Although Sertoli cells are thedirect targets of MEHP, the primary conse-quence of exposure to rodents is a markedincrease in germ-cell apoptosis (Richburg andBoekelheide, 1996).

Phthalates undergo rapid metabolism, andshare PA as a common metabolite, in addi-tion to forming their own specific metabolites

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

(Albro et al., 1987) (Figure 3). Albro andThomas (1973) reported that PA was one ofthe metabolic products excreted in urine whenDEHP was administered orally to rats. PA, whenadministered orally to rats, is neither apprecia-bly metabolized nor retained in the organs ortissues (Williams and Blanchifield, 1974). Limet al. (2007) found that the terminal elimina-tion half-life (t1/2) of PA was 5–7 h. Treatment ofadult male Swiss albino mice with PA resultedin induction of dominant lethal mutations andinduced dose-dependent increases in abnor-mal sperm during meiotic and postmeioticstages of spermatogenesis. The results obtainedindicate that PA is a germ-cell mutagen (Jhaet al., 1998). PA isomers are suspected to bepotent androgen receptor antagonists and mayproduce abnormalities in the male reproduc-tive system (Japan Environment Agency [JEA],1998; Sharpe, 1998). Pavan et al. (2001)noted that molecular mechanisms underlyingPA and steroid-hormone responses in the WISHcell were associated with estrogen-receptorbinding.

There were no adverse effects on fetaldevelopment in mice administered a singleinjection of OPA during pregnancy. There wereno evidence of OPA-mediated carcinogenicity

in chronic feeding studies in rats and mice, andno mutations were detected in bacterial tests(BIBRA Working Group, 1989).

Our in vitro study showed that directcytotoxicity induced by PA isomers was notlikely to be the cause of adverse effectsobserved in testes of affected animals. Cell via-bility was assessed in TM4, NTERA 2 cl. D1,NCTC clone 1469, and FL 62891 cells usingthe MTT assay, but no significant cytotoxicywas observed in NCTC clone 1469 and FL62891 cells. However, cytotoxicity was notedat various concentrations of PA isomers inTM4 and NTERA 2 cl. D1 cells. The testicularcells appeared to be more sensitive to toxicantsthan are liver cells. In addition, PA-inducedcytotoxicity was more potent than IPA, TPA,and OPA.

The animal experiment in this study wasused to explore relationships between CASAparameters and PA isomers. Although resultswere not statistically significant except ata PA concentration of 1000 mg/kg/d, anoverall pattern of decline was observed inCASA parameters VSL, VCL, and LIN forphthalic isomers PA, IPA, TPA, and OPA. Theabsence of consistent, statistically significantdose-response relationships may reflect a lack

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of power due to the relatively small number ofanimals.

Several studies demonstrated a relation-ship between CASA parameters and chemicalexposure. Epichlorohydrin, after 4-h inhala-tion exposure, transiently reduced path velocitydespite no significant change in percent motilesperm (Slott et al., 1990). Alpha-chlorohydrinadministered to male hamsters for 4 d resultedin a significant dose-dependent decline in VCL,VAP, and VCL, despite no change in percentmotile sperm. In addition, alpha-chlorohydrinexposure was associated with a nonlinearimpairment for in vitro fertilizing ability, whichexhibited a threshold-like response. Jelks et al.(2001) examined alpha-chlorohydrin (ACH) atdifferent concentrations (5, 10, 25, 50, and75 mg/kg, po) in rats, and effects on spermATP levels, sperm motility, and ability of spermto bind and penetrate rat oocytes. Computeranalysis of sperm motility indicated that VSLwas the most sensitive parameter to ACH treat-ment; VSL significantly decreased in rat sperm3 h after exposure (25 mg/kg). Data suggest thatCASA parameters may serve as a more sensi-tive marker of reproductive toxicity than semenparameters (Perreault and Cancel, 2001). Onemechanism by which sperm motion or testesmay be impaired includes oxidative stress,the production of reactive oxygen species(ROS), and subsequent lipid peroxidation ofsperm plasma membrane although the prob-able mechanisms may depend on type ofchemicals (Storey, 1997; Armstrong et al.,1999; Martinez et al., 2014; Almášiová et al.,2014).

Ono et al. (1999) examined the associa-tion between toluene levels and epididymalsperm counts, sperm motility, and sperm qual-ity. Data demonstrated that high concentra-tions of toluene might directly target spermin the epididymis and disrupt sperm matura-tion. Selevan et al. (2000) also examined theassociation between air pollution levels andVSL, VCL, and LIN, and found that mediumlevels of air pollution adversely affected VCL,but improved LIN. Adamkovicova et al. (2012)reported that cadmium and diazinon admin-istration to rats led to a significant increase

in ALH and significant fall in BCF by usinga CASA system. Although CASA parametersmay prove to be sensitive biomarkers of repro-ductive toxicity in vivo, these are difficult tocompare across studies because of the use ofdifferent CASA instruments and settings (Daviset al., 1992; Castellini et al., 2011; Mortimerand Mortimer, 2013). Despite this limitation, invivo studies demonstrated that CASA param-eters may be used to predict fertility (Younet al., 2011; Fréour et al., 2012; Broekhuijseet al., 2012) and pregnancy (Macleod andIrvine, 1995; Larsen et al., 2000; Isobe, 2012).Further, epidemiologic studies have used CASAparameters as a marker of altered semen qual-ity (Mukhopadhyay et al., 2010; Vested et al.,2011).

The concentration of PA in biological sam-ples may be useful as a sensitive biomarker ofreproductive toxicity, or as an indirect indica-tor to estimate prevalence of total exposure tophthalates and marker of altered semen qual-ity and mobility. In vitro cell viability assaysshowed that TM4 and NTERA 2 cl. D1 cellswere more sensitive to PA and OPA than NCTCclone 1469 and FL62891 cells. Data suggestthat PA and PA isomers demonstrated specificand significant in vitro and in vivo reproductivetoxicity, specifically, sperm toxicity and testiscell cytotoxicity. Of the PA isomers, PA seemsto be the most toxic and may serve as a surro-gate biomarker for reproductive toxicity uponmixed 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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