In Vitro Evaluation of Plasticizer Activity on the Growth and Metabolism of Chick Embryo Fibroblasts and on the Development of Chick Embryo Lungs G

STABELLINll ,

0

FIOCCH/2,

A

PELLA TIl AND

A

CARUSO l

'Institute of Histology and Embryology, University of Ferrara, Ferrara, Italy; 2Nephrology Unit, Unita Sanitaria Locale 31, Ferrara, Italy

Administration of di[2-ethylhexyl) phthalate (DEHP) to primary cultures of chick embryo fibroblasts brought about a decrease in cell proliferation rate after 48 h and an inhibition of both DNA and protein synthesis measured by pHlthymidine and pH]leucine, respectively, after 48h. The growth of chick embryo lung rudiments in vitro was also depressed by DEHP treatment. Lung rudiment were smaller in DEHP-treated embryos after 6 days' treatment. These results indicate that DEHP has a cytostatic effect on embryonic cells and tissues. L'administration de di{2-ethylhexyl) phthalate (DEHP) a des cultures primaires de fibroblastes d'embryon de poulet a provoque une diminution du taux de proliferation cellulaire apres 48 heures et une inhibition de la synthese a la fois d' ADN et de proteines mesuree, respectivement, par pHlthymidine et pH]leucine apres 48 h. La croissance des ehauches pulmonaires de l'embryon de poulet in vitro a egalement ete abaissee par Ie traitement DEHP. Les ebauches pulmonaires etaient plus petites dans les embryons

475

traites au DEHP apres un traitement de 6 jours. Ces resultats indiquent que Ie DEHP a un effet cytostatique sur Ies celluies et tissus embryonnaires. KEY

WORDS:

Dr(2-ETHYLHEXYL)

PHTHALATE;

EMBRYO;

FIBROBLASTS; LUNG; GROWTH; DEVELOPMENT

INTRODUCTION Medical devices made of plastic have greatly improved in recent years and now polyvinyl chloride is widely used in a variety of medical fields such as in dialysis equipment. Materials derived from polyvinyl chloride may be treated with plasticizers to render them soft and pliable; however, the plasticizers are not chemically bound to the polymer and may leach out from the plastic material. These plasticizers are not stable and may break down into their constituent components, such as di(2-ethylhexyl) phthalate (DEHP) and, in patients undergoing haemodialysis, these components may enter the body.' - 2 Of the total weight of a medical haemodialysis device, DEHP may represent approximately 20 - 40%. Phthalates have been detected in the organs of two patients who died after receiving transfusions of blood that had been stored in DEHP-treated plastic bags." As a result of these findings, many studies have assessed the toxic effects of phthalates and it has been observed that they can induce liver tumours in rats' and cause a proliferation of mitochondria' and peroxisomes" in liver cells. Thickening of the peritoneal membrane and alterations in the capacity of ultrafiltration have also been described in patients treated by continuous ambulatory peritoneal dialysis for long periods of time.' The toxicity of DEHP on embryonic cells

476

remains largely uninvestigated and, therefore, the present study was carried out to assess the effects ofDEHP on cell proliferation and DNA and protein synthesis in primary cultures of chick embryo skin fibroblasts. The effect of DEHP on the developmental process in organotypic cultures of chick embryo lung rudiments was also studied.

MATERIALS AND METHODS MONOLAYER CULTURES Back skin fragments were removed under sterile conditions from 6-day old chick embryos staged according to Hamburger Hamilton table," cut into small pieces and dissociated in 0.25% trypsin (Difco Laboratories, USA) in calcium- and magnesium-free Hearle balanced salt solution at room temperature for 30 min. The dissociated cells were filtered through a nylon mesh, centrifuged at 35 g for 10 min, washed with phosphate-buffered saline (pH 7.4) and suspended in medium 199 (Gibco, USA) plus 10% foetal calf serum and 100 IU/ml penicillin and 10 ug/ml streptomycin. Aliquots, of cell suspension (106 cells/ml) were plated in plastic flasks or multiwell plates (Nunc, Denmark) in a humidity-saturated atmosphere (5% carbon dioxide at 37°C).After plating 24 h, the medium was substituted either by medium 199 plus

ORGANOTYPIC CULTURE

10 ug/rnl dimethyl sulphoxide (control cultures) or by medium 199 plus 10 ug/rnl DEHP in dimethyl su l ph oxide (treated cultures). All the cell cultures were maintained in vitro for 24 or 48 h. After treatment, cell viability was measured by the ability of the cells to exclude trypan blue."

Lungs were removed under sterile conditions from 6-day old chick embryos (Selice Incubator Co., Italy), staged according to the Hamburger - Hamilton table," and cut into right and left halves. Each half was placed in a culture dish on testacea membrane in the presence of suitable culture medium and incubated for 6 days at 37°C. Using a slightly modified method to that of Wolffand Haffen, 11 semi-solid natural media were employed. The standard nutrient consisted of 1% agar (Difco, USA) in Gey's balanced saline, foetal calf serum (Gibco, USA) and medium 199 in a ratio of 6 : 3: 4 plus and 100 IU/ml penicillin and 10 ug/rnl streptomycin. Treated cultures were incubated for 24 or 48 h with standard previously nutrient containing 10 ug/rnl DEHP. Cultures were observed daily and photographed under a Leitz stereomicroscope at x 30 magnification. Explants were fixed in Bouin fluid and routine histological procedures were carried out. Serial sections of 5 um were cut at intervals of 100 urn and stained for morphological examination with haematoxylin/eosin. Morphogenesis was evaluated by counting the second and third subdivisions of bronchial branching under a stereomicroscope. Random controls were also performed on histological slides.

CELL GROWTH At appropriate time intervals (24 or 48 h), cells were counted using crystal violet staining in four-well culture plates in situ according to the method of Gillies et 01. ' 0

INCORPORATION OF f3H]THYMIDINE Control and DEHP-treated cells maintained in vitro for 48 h after the plating were pulse labelled with 1.0 u Ci [3HJthymidine (20 Ci/rnrnol: New England Nuclear, FRG) during the last 3 h of the culture period. Cells were solubilized with 0.5 M sodium hydroxide and an aliquot was precipitated with 10% trichloroacetic acid for 30 min at 4°C, filtered on glass microfibre filters (GFIC; Whatman, UK) and washed at room temperature with 5% trichloroacetic acid maintained at O°C in an ice bath. The filters were then dried and counted using 10 ml scintillation counter (trichloroacetic acidinsoluble fraction). Total cell radioactive uptake was measured in separate aliquots of solubilized cells counted using 10 ml scintillation fluid and the results obtained were expressed as decompositions per minute per 10 6 cells [dpm/f.O" cells).

STATISTICAL ANALYSIS Means (± SD) were obtained from four parallel cultures and Student's t-test was used to compare the results for control and DEHPtreated cultures.

INCORPORATION OF [3H]LEUCINE Control and DEHP-treated cells cultured for 48 h after plating were pulse labelled with 1.0 p Ci [3H]leucine (52 Ci/mmol; New Nuclear England, FRG) during the last 3 h of the culture period and then processed as described for incorporation of [3H[thymidine. The results obtained were expressed as dpm/LO" cells.

RESULTS As shown in Fig. 1, administration of DEHP did not greatly alter the rate of cell division during the first 24 h of treatment; however, after 48 h the cell numbers per cultures were reduced in the presence of DEHP. The addition of dimethyl sulphoxide to the

477

1.3

------- Control

1.1

_______ DEHP

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03 .D

E ::J c Cii

0.9

0.7

o

0.5

0.3

24

0

48

Time (h)

Effect of 10 pg/ml di(2-ethylhexyl) phthalate (DEHP) on the growth of chick embryo fibroblast. The experiments were performed four times with similar results; data shown are from a single representative experiment.

Mean (:1: SD) of fHjthymidine incorporation in chick embryo fibroblast cultures treated with 10 pglml di(2-hexylethyl) phthlate (DEHP) for 48 h; 1.0 pCi fHjthymidine (20 Ci/mmol) was added during the last 3 h

Culture

Total radioactivity (dpm/10 6 cells)

Control

367840 ± 12455

DEHP

373912 ± 18731

P-value

Acid-insoluble fraction (dpm 106 cells)

P-value

247821 ± 10635 NS

dpm, decompositions per minute.

478

175412 ± 12399

< 0.01

Mean (± SD) of pH]leucine incorporation in chick embryo fibroblast cultures treated with 10 pg/ml di(2-hexylethyl) phthlate (DEHP) for 48 h; 1.0 pCi pH}/eucine was pulsed during the last 3 h

Culture

Total radioactivity (dpm/10 6 cells)

Control

60124 ± 5122

DEHP

39347 ± 3 861

P-value

Acid-insoluble fraction (dpm 10 6 cells)

P-value

21 277 ± 3 184 < 0.01

8181 ± 1 645

< 0.01

dpm, decompositions per minute.

detected in biological fluids stored for a few months in plastic bags (Stabellini G, unpublished results). The data show that DEHP at a concentration of 10 ug/rnl inhibited both DNA and protein synthesis in cultured chick embryo fibroblasts. Administration of DEHP also brought about a strong decrease in both ['H]leucine uptake and its incorporation into proteins. The decrease in DNA synthesis was the result of a reduced incorporation of [3H]thymidine in the DNA, whereas uptake of [3H]thymidine was not affected. These data suggest that administration ofDEHP primarily brings about inhibition of protein synthesis, which in turn depresses DNA duplication and then cellular proliferation. Jacobson et a1. 12 showed an impairment of cell proliferation, measured as ['HJthymidine incorporation, in human fibroblasts grown in the presence of serum that had been stored for 3weeks in DEHP-treated polyvinyl chloride bags. Storage of peritoneal liquid in polyvinyl chloride plus DEHP bags has also been shown to result in a decrease in protein synthesis in L-929 cells," and reduced incorporation of [3H]leucine has been observed in cells grown in presence of mono 2-

culture media did not influence the cell growth or any other evaluated parameters (data not shown). Treatment with DEHP for 48 h decreased the incorporation of ['H]thymidine into the newly synthesized DNA to 71% ofthe control value, whereas the total radioactivity was unchanged (Table 1). Treatment with DEHP for 48 h brought about a decrease in both the total cellular radioactivity (-34.6%) and the radioactivity incorporated into the protein (-61.6%) in respect of [3H]leucine incorporation in chick embryo fibroblast culture (Table 2). The growth of chick embryo lung in vitro was also influenced by DEHP. Lung rudiments cultured in presence of DEHP for 6 days were smaller than the controls (Fig. 2) and the number of bronchi per section was reduced (-22.8%) in DEHP-treated chick lung cultures (Table 3).

DISCUSSION The effects of DEHP on embryonic cells were studied using concentrations similar to those

479

G Stabellini, () Fiocchi, 11 Pellaii et al.

Plasticizer activity on chick embryo cells

FIGURE 2

Chick lung explants maintained for 6 days in (A) control medium and (8) In 10 pg/ml dl(2-ethylhexyl) phthalate (DEHP) containing medium; photographed under stereomlcroscope (x 45 magnification).

480

Bronchial branching of chick embryo lung cultures treated with 10 pglml di(2-hexylethyl) phthlate (DEHP) for 6 days

Culture

No. of cultures

Second and third subdivisions per culture

Control

12

17.64 ± 1.92

DEHP

12

13.61 ± 1.175

ethylhexyl) phthalate, which is a metabolite of DEHP.14 The data obtained in the present study are consistent with those previously reported and, in this study, a correlation was detected between the decrease in cellular biosynthesis and the impairment of cell growth in DEHP-treated cultures. The data also showed that administration of DEHP reduced cell proliferation in organ culture. A decrease in the number of bronchial trees and a reduction in the size of embryonic

P-value

< 0.01

chick lung cultured in presence ofDEHP was apparent, leading to the conclusion that DEHP has a cytostatic effect on embryo cells and tissues in vitro. Developmental events, such as the formation of bronchial rudiments, are delayed but not completely inhibited.

ACKNOWLEDGEMENT This work was supported by 91/60106/071 MURST (Italy) grant.

REFERENCES Science 1970; 170: 460. 4 US Department of Health and Human Services: Carcinogenesis bioassay of di-2ethylhexyl phthalate in F344 rats and B6C3Fl mice. National Toxicology Program, NIH Publication No. 82 - 1773. Bethesda: National Institutes of Health, 1982. 5 Hashimoto T: Individual peroxisomal ~­ oxidation enzymes. Ann NYAcad Sci 1982; 386: 5 - 12. 6 Crane I, Zamattia J, Masters CJ: Alteration

1 Naessberger L, Arbin A, Oestelius J: Exposure of 'patients to phthalates from polyvinyl chloride tubes and bags during dialysis. Nephron 1987; 45: 286 - 290. 2 Jaeger RJ,Rubin RJ:Migration of a phthalate ester plasticizer from polyvinyl chloride blood bages into stored human blood and its localization in human tissues. N Engl J Med 1972; 287: 1114 - 1118. 3 Jaeger RJ, Rubin RG: Plasticizers from plastic devices: extraction, metabolism, and accumulation by biological systems.

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in the integrity of peroxisomal membranes in livers of mice treated with peroxisome proliferators. Mol Cell Biochem 1990; 96: 153 - 161. Oreopoulos DG: Peritoneal membrane: handle with care. Peritoneal Dialysis Bull 1983; 111: 113 -115. Hamilton HL: Lillie's Development of the Chick: an Introduction. New York: H Holt 1952. Patterson MK Jr: Measurement of growth and viability of cells in culture. Methods Enzymol1979; 58: 141 - 145. Gillies RJ, Didier N, Denton M: Determination of cell number in monolayer cultures. Anal Bioehem 1986; 159: 109 113. Wolff E, Haffen KC: Sur une methode de culture d'organes embryonaires in vitro. Tex Rep Biol Med 1952; 10: 463 - 472. Jacobson MS, Kevy SV, Parkaman R, et ol: An in vitro evaluation of a new plasticizer

for polyvinyl chloride medical devices. Transfusion 1980; 20: 443 - 447. 13 Wieslander AP, Nordin MK, Kjellstrand TT, et al: Toxicity of peritoneal dialysis fluid on cultured fibroblasts, L-929. Kidney Int 1991; 40: 77 - 79. 14 Thysen B, Morris PL, Gatz M, et al: The effect of mono(2-ethylhexyl phthlate on Sertoli cell transferin secretion in vitro. Toxieol Appl Pharmacol1990; 106: 154 157.

G Stabellini, 0 Fiocchi, A Pellati and A Caruso In Vitro Evaluation of Plasticizer Activity on the Growth and Metabolism of Chick Embryo Fibroblasts and on the Development of Chick Embryo Lungs The Journal of International Medical Research 1992; 20: 475 - 482 Received for publication 7 September 1992 Accepted 17 September 1992 © Copyright 1992 Cambridge Medical Publications

Address for correspondence DR

G

STABELLINI

Istituto di Istologia ed Embriologia, via Fossato di Mortara 64, 44100 Ferrara, Italy.

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In vitro evaluation of plasticizer activity on the growth and metabolism of chick embryo fibroblasts and on the development of chick embryo lungs.

Administration of di(2-ethylhexyl) phthalate (DEHP) to primary cultures of chick embryo fibroblasts brought about a decrease in cell proliferation rat...
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