Mutation Research, 262 (1991) 85-92 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0165-7992/91/$ 03.50 ADONIS 016579929100002C

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MUTLET 0451

In vitro genotoxicity of dimethyl terephthalate

S. Monarca 1, B.L. Pool-Zobel 2, R. Rizzi 3, P. Klein 2, P. Schmezer 2, E. Piatti 3, R. Pasquini 4, R. De Fusco 5 and D. Biscardi 6

1Chair of Environmental Health, School of Medicine, University of Brescia, Brescia (Italy), 2German Cancer Center, Heidelberg ( F.R.G.), 3Chair of Pharmacology, School of Medicine, University of Milan, Milan (Italy), 4Department of Hygiene, University of Perugia, Perugia (Italy), 5Department of Experimental Pharmacology and 6Department of Environmental Physiology, University of

Naples, Naples (Italy)

(Received I May 1990) (Revision received 18 September 1990)

(Accepted 21 September 1990)

Keywords: Dimethyl terephthalate; In vitro genotoxicity; Ames test; DNA damage; Chromosome aberrations in human lymphocytes; Selective DNA amplification

Summary

Dimethyl terephthalate (DMTP), the para configuration of dimethyl phthalate, is one of the basic monomers used in the synthesis of polyethylene terephthalate (PET) plastics. Human exposure to DMTP may primarily occur during the manufacture of PET fibers and films. The mutagenic potential of dimethyl terephthalate was evaluated using a battery of in vitro short-term tests: the Ames test; DNA single-strand break assays in CO60 cells and in primary rat hepatocytes; UDS in HeLa cells; chromosome aberration and micronucleus assays in human peripheral blood lymphocytes; selective DNA amplification in CO60 and in Syrian hamster embryo cells. The results of this battery of in vitro assays clearly show that DMTP is non- genotoxic. By contrast, other authors have found DMTP to be an in vivo clastogenic compound and sug- gested that the mechanisms involved in these in vivo effects seem to have nothing in common with genotoxici- ty and are still unknown.

Phthalate esters are widely used in the manufac- ture of plastics and in many other chemical pro- cesses and may be detected in all ecosystems (Douglas et al., 1986). Health concern has been

Correspondence: Dr. S. Monarca, Chair of Environmental Health, School of Medicine, University of Brescia, Brescia (Italy).

raised for the possible toxic effects of these ubi- quitous pollutants. In fact, some phthalate esters have been found to cause malignant tumors in mice and rats (NCI, 1979; Warren et aI., 1982; Kluwe et al., 1982; Kluwe, 1986; Schmezer et al., 1988), to have tumor-initiating and -promoting effects (Ward et al., 1986), and to possess teratogenic/ fetotoxic (Tomita et al., 1982; Shiota and

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Nishimura, 1982) and mutagenic activities (Kozumbo et al., 1982; Seed, 1982; Tomita et al., 1982; Douglas et al., 1986; Phillips et al., 1986; Goncharova et al., 1988; Schmezer et al., 1988). Dimethyl terephthalate (DMTP), the para con- figuration of dimethyl phthalate, is one of the basic monomers used in the synthesis of polyethylene terephthalate (PET) plastics. Human exposure to DMTP may primarily occur during the manufac- ture of PET fibers and films, and potentially, as recently suggested, also by consumption of food stored in PET containers (De Fusco, 1990). In par- ticular De Fusco et al. (1990) have found leaching of unknown mutagenic substances from PET bot- tles into mineral water. Studies on the in vitro genotoxicity of DMTP are very limited: DMTP has been tested with the Salmonella typhimurium mutagenicity ( 'Ames') test and was found to be negative (Kozumbo et al., 1982). However, recent- ly DMTP was found to be an in vivo clastogenic agent in a mouse micronucleus test (Goncharova et al., 1988). DMTP is also one of the compounds capable of inducing calculi in the bladders of ex- perimental animals and many of these compounds also induced bladder cancer during chronic feeding studies (Heck, 1987).

In order to better study the in vitro genotoxicity of DMTP, we have tested it using a series of in vitro short-term tests: the Ames test; DNA single-strand break assays in CO60 cells and in primary rat hepatocytes; unscheduled DNA synthesis (UDS) in HeLa cells; chromosome aberration and micronucleus assays in human peripheral blood lymphocytes; selective DNA amplification in CO60 and Syrian hamster embryo cells.

Materials and methods

Dimethyl terephthalate (DMTP) (CAS No. 12-06-16) was obtained from the largest Italian PET plastic industry, which was also the source of the PET bottles for carbonated drinks used in our previous leaching study (De Fusco et al., 1990). Since DMTP is practically insoluble in water, it was dissolved by sonication in a mixture of DMSO and Tween 20 (29:1) and this solution was studied by

means of the following tests at increasing doses.

Mutagenicity in S. typhimurium DMTP was tested in strains TA98, TAI00,

TAI02, TA1535, TA1537, TA1538 of S. typhi- murium for reversion to histidine prototrophy at increasing doses (up to 5 mg/plate), with and without metabolic activation ($9), as described by Ames et al. (1975) and Maron and Ames (1983).

DNA single-strand breaks in primary hepatocytes Hepatocytes were isolated from rats via the 2-

step in situ perfusion technique described by Bradley et al. (1982), with slight modifications (Romruen and Pool, 1984) and the test was per- formed according to previously reported methods (Bradley and Sina, 1984; Kohn et al., 1981). Cells (2 × 10 6) suspended in 1 ml medium were incubated for 1 h at 37°C with different concentrations of DMTP. After the incubation, 0.25-ml aliquots were taken for analysis of cytotoxicity, via the trypan blue exclusion test. The remaining 1.5 × l0 6

cells were lysed on filters to isolate DNA which was then eluted at alkaline pH according to the pro- cedure of Kohn et al. (1981) and Pool et al. (1990). The DNA in the collected fractions and that re- maining on the filter was measured fluorometrical- ly using the Hoechst 33258 dye. Calculations of the °7o DNA retained on the filter during the elution process were performed. As evaluation, the °7o DNA retained on the filter after 18 h is shown. A clearly positive effect is evident when the °7o DNA retained on the filter in the control (C) minus °7o DNA retained in the treatment groups (T) is > 20 (C-T>20OT0), at non-toxic doses (viability test /control > 70°70).

DNA single-strand breaks in C060 cells This assay was carried out in SV40-transformed

Chinese hamster embryo cells (CO60 line) as described for the hepatocytes (see above).

Unscheduled DNA synthesis (UDS) in HeLa cells DMTP was tested for its ability to induce

unscheduled DNA synthesis in HeLa cells, ac- cording to the method of Bianchi et al. (1982). For

treatments, 2.5 × 10 6 HeLa cells were seeded into 5 ml of Eagle's minimal essential medium sup- plemented with 10o70 calf serum (MEM) in petri dishes (6 cm diameter) containing 4 cover-slips (12 mm x 12 mm), and incubated for 24 h at 37°C. The cultures were rinsed 3 times with PBS, and in- cubated at 37°C for 1 h with the desired concentra- tions of DMTP. Tests with $9 liver homogenates f rom Aroclor-induced Sprague-Dawley rats (0.5 ml/ml) were also performed. At the end of treat- ment the cultures were rinsed 3 times with PBS and incubated with or without hydroxyurea (HU) in MEM. After 15 min tritiated methylthymidine was added to give 10/zCi/ml (2/zCi/mM), and the in- cubation was continued for 3 h at 37°C. After in- cubation the slides were washed twice with cold PBS and then were treated with 3 ml HC1 for 20 min. The slides were washed twice with ethyl ether-ethanol ( l : l ) , twice with cold ethyl ether and then dried. The slides were put into vials, treated overnight with Soluene 350 (Packard), supplied with 10 ml Dimilume 30 (Packard) and then counted for radioactivity in a Packard scintillation counter. Radioactivity counting gives a measure of the thymidine incorporation in each of the follow- ing experimental series: controls (C), controls plus HU (CHU), cultures treated with DMTP (T), and treated cultures plus HU (THU). The inhibition of DNA replicative synthesis by HU in control and treated cultures was indicated by the ratios C H U / C and T H U / T , respectively, and the inhibition due to DMTP by the ratio T /C. The relative increase of DNA radioactivity in the presence of HU ( T H U / T : C H U / C ) , taken as an index of the induc- tion of DNA repair synthesis, was also measured.

Chromosome aberration assay and micronucleus test in human peripheral blood lymphocytes

Intravenous blood was collected in heparinized tubes from 2 non-smoking healthy volunteers (male, 25 years old; female, 28 years old), free f rom pharmacological treatment, and without chromosome fragility and recent viral infection (Preston et al., 1981). The blood cells (1 ml of whole blood) were treated with DMTP in the Go stage of the cell cycle for 4 h at 37°C. The

87

substance was tested at doses of 50-500 /~g/ml (final volume 10/~1). DMSO and bleomycin were used as negative and positive controls, respectively. After washing, 0.5 ml of cell suspension was added to 4.5 ml RPMI 1640 medium, supplemented with 2007o fetal calf serum (FCS) plus antibiotics and 0.13 ml phytohemagglutinin (PHA) and the cultures were incubated at 37°C for 72 h. 0.05 ~g/ml of Colcemid was added to the cultures, 2 h before harvesting. The lymphocytes were harvested and slides were prepared according to Moorehead et al. (1960). Then the slides were coded and stain- ed with 4°7o Giemsa solution.

0.5 ml of the heparinized whole blood was treated with DMTP solution (50-500 #g/ml) and was cultured in RPMI supplemented with 20°7o FCS, antibiotics and P H A (Fenech and Morley, 1986). Cytochalasin B (Sigma, 3/~g/ml) was added after 44 h and the cultures were grown at 37°C and sampled at 72 h. After a mild hypotonic treatment, the cells were fixed and dropped onto the slides, which were coded, air-dried and stained with 4°70 Giemsa solution. The micronucleus scoring was performed on 1000 binucleated cells per subject.

Selective DNA amplification in AAV2-infected Syrian hamster embryo cells

We studied the selective DNA amplification in primary Syrian hamster embryo ceils (SHE) in- fected with adeno-associated virus (AAV type 2) according to Yalkinoglu et al. (1988) and Klein et al. (1990). This system, where the DNA of infecting AAV is used as a marker for DNA amplification due to genotoxic compounds, is applicable to a variety of different target cells and is not restricted to transformed or SV40 DNA-containing cells, as was originally described (Lavi, 1981). Here, the target cells derived from Syrian hamster embryos were isolated according to Pienta (1980).

Selective DNA amplification (SDA) was deter- mined according to Lavi (1981) and Pool et al. (1989). AAV-infected Syrian hamster embryo cells were plated in a tissue culture flask ( 5 x 1 0 s cells/flask, 2 flasks per concentration of com- pound) containing 5 ml Dulbecco's modified essen- tial medium supplemented with 10°70 fetal calf

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TABLE 1

MUTAGENICITY OF DIMETHYL TEREPHTHALATE (DMTP) in Sulmonella typhimurium

DMTP

&g/plate)

Revertants/platea

TA98 TAlOO TA102 TA1535 TA1537 TA1538

- s9 is9 -s9 + s9

0 18*2 27+ 2 60+ 6 71+5

0.5 19k-3 21t 5 63+ 2 83+2

5.0 15k2 26kll 52+ 2 77+3

50.0 16t 1 19+ 5 64+_11 14+5

500.0 16k3 23k 4 71+ 9 60+7

5000.0 11*2 19+ 3 57+13 77+9

-s9 is9

115+7 181+49

115*9 211* 4

109k7 179+26

104+_3 185+22

99+4 140t26

86+5 153+ 9

-s9 is9 -s9 + s9

4+1 17*5 4*3 lOk2

7t1 18k3 8+1 6*1

4+1 17*7 3k2 8+2

3k2 16+5 4k2 6+2

7+2 14+2 6k4 4+1

8kl 13+3 5k2 0

-s9 +s9

8+2 31+4

11*5 33k4

7*3 27 + 3

10*2 34+5

9+3 28+2

16+2 16k2

a Mean values of 3 independent experiments using triplicate plates

serum and penicillin/streptomycin. The compound was tested in wells of a microtiter plate containing a total of 24 wells (1 x lo5 cells/3 ml medium). After 24 h the cells were treated with the test com- pound dissolved in 10 ~1 DMSO. After 4 days of in- cubation, cells were harvested, counted and viabili- ty was determined by trypan blue exclusion. The AAV DNA content was detected by in situ hydridization. A survival index (SI) based on 100% survival of cells in the solvent control was calculated according to: SI = % survival of the treated cells x lOO/% survival of the cells in con- trol. An amplification factor (AF) was calculated as cpm in the filters of the treated group divided by cpm in the filters of the control. Using these factors a genotoxicity index (GI) was calculated for evalua- tion of the results: GI = (AFx SI)/lOO. Accor- dingly, the results were determined as positive or negative as follows: AFG2.9, - ; 3-10.9, + ; 1 l-25, + +; >25, + + + . AF values were only valid when the viability of the treated cells (reflected in GI) was not reduced by more than 30% as compared with the viability of the control cells. In addition, we also determined the total number of cells harvested, as this parameter is of biological importance for selective DNA amplifica- tion.

Results

Ames test All the dose levels assayed (from 0.5 to 5000

pg/plate) were found to be negative in the Ames test, with all the strains, with or without metabolic activation (Table 1). Evidence of toxicity was observed at the highest dose levels in TA98 ( - S9) and TA1537 (+ S9).

TABLE 2

ASSAY FOR THE INDUCTION OF DNA SINGLE-STRAND

BREAKS IN PRIMARY RAT HEPATOCYTES

Expt. Compound

No. (amolekube)

Relative

viability

(070)

C-T

(To)”

1 DMTP

0

3.75

7.50

15.00

NMBAb

3.13

100

79 3.5

94 7.5

88 10.4

103 50.4

2 DMTP

0 100

3.75 88 10.7

7.50 85 14.7

15.00 100 15.2

NMBAb

3.13 79 42.8

a C-T, % DNA retained on filtertest - % DNA retained on

filter con,rot; means of 3 determinations per concentration.

b NMBA, N-nitrosomethylbenzylamine (positive control com-

pound).

TABLE 3

DETERMINATION OF UNSCHEDULED DNA SYNTHESIS (UDS) in HeLa CELLS BY DMTP a

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Compound $9 Incorporation of tritiated T H U / T CHU/C

thymidine a

- H U + H U

(cpm _+ SD) (cpm _+ SD)

T H U / T : C H U / C

DMTP

(#g/ml)

0 - 28,673 ± 1,939 792 ± 76 0.0276 1.0000 1.0000

+ 43,070± 3,857 1,460 ± 385 0.0338 1.0000 1.0000

5 - 17,726 ± 4,904 558 ± 88 0.0314 0.7045 1.1376

+ 40,241 ± 1,117 1,057 -+ 254 0.0262 0.7239 0.7751

50 - 4,858± 1,056 160-+ 14 0.0329 0.2020 1.1376

+ 35,520±2,973 767-+ 94 0.0215 0.5253 0.6360

500 - 3,762± 731 143 ± 51 0.0380 0.1805 1.3768

+ 32,757 -+ 3,235 805 -+ 195 0.0245 0.5513 0.7248

5000 - 3,043-+ 359 128-+ 33 0.0420 0.1616 1.5217

+ 24,975 ± 2,233 618 ± 179 0.0247 0.4232 0.7307

EMS b

(100mM) - 607± 222 561 -+ 88 0.9242 0.7083

MNNG c

(50 mM) + 6,635-+2,593 4,425-+ 1,913

33.4855

0.6669 3.0308 19.7308

a Mean values of 2 experiments (see text for explanations).

b EMS, ethyl methanesulfonate.

c MNNG, N-methyl-N'-nitro-N-nitrosoguanidine.

TABLE 4

CHROMOSOME ABERRATIONS IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES CULTURED IN VITRO

Compound

(#g/ml)

Abnormal metaphases (%)a Chrom. aberr./cell

Male Female Male Female

subject subject subject subject

DMTP

0 4 2 1 1

50 3 2 1 1 100 5 3 1 1

250 4 4 1 1

500 3 4 I 1

Bleomycin

50 19 15 5.4

a 100 metaphases/dose/subject.

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DNA single-strand breaks in C060 cells and in primary hepatocytes

DMTP produced DNA damage neither in primary rat hepatocytes (Table 2) nor in CO60 cells (results not shown).

UDS in HeLa cells

The results of the UDS tests are reported in Table 3. DMTP was negative when assayed in this system at 5-5000 #g/ml, both with and without metabolic activation.

Chromosome aberration assay and micronucleus test in human peripheral blood lymphocytes

We analyzed 100 metaphases per dose per sub- ject. The frequency of chromatid and chromo- somal gaps (achromatic lesions) and breaks in lym- phocytes was not significantly different from the controls, as determined using the chi-square test (Table 4). Only in the positive control (bleomycin) were rings, dicentrics, acentric fragments, and dou- ble minutes present in addition to gaps and breaks. The mean number of aberrations per cell was 1 for all the cell samples; it was > 1 only in the positive control. The difference in frequency of micronuclei between the treated samples and controls was not significant (Table 5); in the treated samples the values ranged from 3 to 8, similar to the spon- taneous micronucleus frequency.

TABLE 5

M1CRONUCLEI IN HUMAN PERIPHERAL BLOOD LYM- PHOCYTES

Compound

(tzg/ml)

Micronuclei/1000 binucleate cells

Male

subject Female

subject

3

3 7

5

8

DMTP

0 4

50 5 100 8

250 7

500 7

Bleomycin

50 62 56

Selective DNA amplification

AAV-SDA was not induced in primary Syrian hamster embryo cells by DMTP (Table 6).

Discussion

In this investigation, we have found that dimethyl terephthalate (DMTP), a widely used precursor of polyester plastics, is non-genotoxic in a series of in vitro short-term tests. In particular, as previously reported by Kozumbo et al. (1982) in a more limited experiment, we did not find DMTP genotoxic in the Ames test, using a more complete set of strains and higher concentrations of the com- pound, tested over a range of non-toxic to partially cytotoxic doses.

DMTP failed to reveal DNA-damaging activity not only in CO60 cells, but also in rat primary hepatocytes. These systems are expected to reveal a broad range of genotoxic compounds, while hepatocytes are considered to be particularly useful for their metabolic activity.

We were unable te demonstrate the induction of UDS in HeLa cells, and of chromosome aberra- tions and micronuclei in human lymphocytes.

TABLE 6

SELECTIVE DNA AMPLIFICATION IN AAV2-INFECTED

SYRIAN HAMSTER EMBRYO CELLS

Compound No. of % Viable SI S cpm AF b

(#g/ml) ceils ( × 10 s)

DMTP

0 6.0 90.9 100 60 1.0

2.5 3.0 100.0 110 16 0.3

5.0 5.4 100.0 110 63 1.0

10.0 8.4 100.0 110 68 1.1 DMBA c

5.0 2.4 75.0 82.5 126 2.1 Mitomycin C

1.8 3.6 83.3 91.6 250 4.2

Daunomycin

0.6 0.6 100.0 110 331 5.5

a SI, survival index.

b AF, amplification factor.

c 1,12-Dimethylbenzanthracene.

Selective D N A ampl i f ica t ion , which appears to

be involved in the processes o f t umor ini t ia t ion and

t u m o r progress ion (Stark and Wahl , 1984; Poo l

and Schm/~hl, 1987), was studied in Syrian hamster

e m b r y o pr imary cells, with negat ive results.

The results o f this bat tery o f in vi tro assays clear-

ly show that D M T P is non-genotoxic . Recent ly it

has been shown that other phthalates , e.g. ,

d i (2-ethylhexyl)phthalate , which are carcinogenic ,

canno t interact with D N A nucleophil ic sites to

f o r m D N A adducts (Lutz, 1986) and an analysis o f

the molecu la r s t ructure has evidenced that

phthala tes , including D M T P , do not possess elec-

t rophi l ic sites (Ashby and Tennant , 1988). This

may explain the negat ive results in in vi tro genotox-

icity tests. By contrast , D M T P and terephthal ic

acid were found to be in vivo clastogenic com-

pounds (Goncha rova et al., 1988). There fore , the

mechan isms involved in these in vivo effects seem

to have noth ing in c o m m o n with genotoxic i ty and

are still unknown. It seems that phthalates can alter

m e m b r a n e s o f cells and the normal processing o f

some, still unde te rmined , intracell structures which

in turn leads to D N A / c h r o m o s o m a l damages

(Phill ips, 1986; Zabre jko and Goncha rova , 1988,

personal communica t ion) .

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Communicated by F.H. Sobels