Toxicology, 17 (1980) 177--182 © Elsevier/North-Holland Scientific Publishers Ltd.

CYTOTOXIC EFFECTS OF AIR POLLUTANTS ON MAMMALIAN CELLS IN VITRO

N.H. SEEMAYER and N. MANOJLOVIC

Medizinisches Inst i tut fiir Umwelthygiene an der Universit~'t Diisseldorf, Gurlittstr. 53, D-4000 Diisseldorf (F.R.G.)

SUMMARY

Extract and fractions of city smog collected from a heavy industrialized area caused in vitro dose-dependent reduction of cell survival of mouse macrophages and a growth inhibition of human fetal diploid lung fibro- blasts. Great differences were observed in both cell systems between the total extract and its different fractions. Cytotoxici ty declined in the se- quence of total extract followed by fractions of cyclohexane, methanol, propanol and of polycyclic aromatic hydrocarbons. Our results illustrate that samples of city smog contain highly toxic substances for mammalian cells in vitro. Differences in cytotoxici ty of particular fractions indicate that toxic agents belong to various classes of chemical substances.

INTRODUCTION

Accelerated industrialization and urbanization, and growing traffic lead to an increase of environmental pollution in most of the highly developed countries. High levels of air pollutants are of particular importance as a hazard impairing human health. Air borne particulate matter or city smog contain a high number of chemical substances, of which carcinogens are also included. Cytotoxic, mutagenic and carcinogenic effects induced by city smog extracts in mammalian cells in vitro have been described [1--3]. We report about a reduction of cell survival of mouse macrophages in vitro and of growth inhibition of human fetal diploid lung fibroblasts in the presence of city smog extracts.

MATERIALS AND METHODS

Cell c u l t u r e s Mouse macrophages of cell line IC-21, developed by Mauel and Defendi

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[4] were cultivated in medium RPMI 1640 supplemented with 10% new- born calf serum as described [5]. Confluent monolayers of IC-21 cells were exposed in medium RPMI 1640 wi thout serum to various concen- trations of city smog extract and its fractions dissolved in DMSO. The final concentrat ion of DMSO in medium was 1%. After 2, 5 and 24 h of incub- ation, cells were detached by t reatment with a trypsin-versene solution and suspended in medium RPMI 1640.

The criterion o f cell survival viability was determined according to Garry and Moore [6] by the dye exclusion test with trypan blue (0.05%) em- ploying a Model 6300 laser Cytograf of Bio/Physics Systems Inc., Mahopac, N.Y. Viability was estimated on 2--4 × l 0 s cells. Human fetal diploid lung flbroblasts were cultivated in Eagle's Minimal Essential Medium (MEM) supplemented with 10% newborn calf serum and antibiotics (Penicillin 100 IU/ml, Streptomycin 100 #g/ml). For analysis of cell growth 250,000 cells were seeded in Falcon flasks of 25 cm 2. Twenty four hours later various concentrations of city smog extract and its fractions dissolved in DMSO (final concentration 1%) were added to cell cultures. The criterion of the cell growth protein content of cell cultures was estimated by the Lowry method and modified according to Oyama and Eagle [7]. Single measure- ments conducted at 0 time and after 24 h, 48 h and 72 h of incubation represent mean values of 2--4 culture vessels.

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Fig. 1. Schematic presentation of fractionation of city smog sample. Total extract (GEX), fraction of methanol (MET), cyclohexane (CYC), polycyclic aromatic hydro- carbons (PAH) and propanol (PRO).

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Collection, extraction and fractionation of city smog Samples of city smog were collected in a heavy industrialized Rhine-

Ruhr area (F.R.G.) using a Draeger-Box-Micron-Filter MB 1700. The ex- traction procedure by organic solvents and ffactionation by column chro- matography according to a schedule developed by Tomingas has been described in detail [8] (Fig. 1). For analysis of cytotoxicity all fractions were quantitatively transferred to DMSO. Besides the total extract (GEX) the following fractions were included in testing: methanol (MET); cyclo- hexane (CYC); polycyclic aromatic hydrocarbons (PAH); and propanol (PRO). Dosage was calculated according to benzo[a]pyrene content, ex- pressed as benzo[a]pyrene-equivalent (BaP-equ.) or of fractions which contained no benzo[a]pyrene (methanol, propanol) as a corresponding volume-equivalent (vol.-equ.).

RESULTS AND DISCUSSION

After incubation periods of 2 and 5 h in the presence of city smog ex- tract (CSE) mouse macrophages revealed a strong reduction of cell survival by cyclohexane fraction at concentrations of 0.8--1.6 pg/ml BaP-equ. (Fig. 2), while other fractions induced only slight effects. After 24 h of incubation, cell survival was strongly reduced by various fractions giving the following series of declining cytotoxicity: total extract (GEX); cyclo- hexane; methanol; propanol; and polycyclic aromatic hydrocarbons (Fig. 3). Total extract, cyclohexane- and methanol-fractions caused a strong re- duction of cell survival at concentrations of 0.2--0.4 pg/ml BaP-equ. or vol.-equ., respectively. In contrast, fractions of propanol and polycyclic aromatic hydrocarbons produced a remarkable reduction of cell survival only at higher concentrations of 0.8--1.6 ~g/ml BaP-equ. or vol.-equ.

In the presence of CSE and its fractions a dose dependent inhibition of growth of human fetal diploid lung fibroblasts was detected, but great differences in the effective dose were observed. Total extract (GEX) and methanol-fraction caused a complete growth inhibition in the presence of 0.2 ug/ml BaP-equ. or vol.-equ., respectively (Fig. 4). In comparison, cyclohexane fraction induced a complete growth inhibition at a concen- tration of 0.8 ~g/ml BaP-equ., while fractions of propanol and polycyclic aromatic hydrocarbons led to a similar effect at higher concentrations of 1.6--1.8 pg/ml. Tissue culture offers a cellular approach to study the mechanism(s) of injury induced by air pollutants. As our results demon- strate, city smog extract and its derived fractions exert, in both cell systems used, a comparable dose-dependent cytotoxic effect in spite of differences of species (mouse, human), cell type (macrophages, lung fibroblasts) and parameters applied (viability, cell growth). Taking into consideration the average concentration of benzo[a]pyrene of 20--200 ng/m 3 air in heavy industrialized areas [9], the effective cytotoxic doses, f.i. of total extract and methanol fractions, are equivalent to 1 m 3 or few cubic meters of air.

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Incubation period: 5 hours ®

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180

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Figs. 4 and 5. Growth inhibition of human fetal diploid lung fibroblasts in the presence of city smog total extract (GEX) and fraction of polycyelic m~omafic hydrocarbon&

So mouse macrophages as well as human lung fibroblasts can be regarded as sensitive indicators of cytotoxicity of city smog.

Further investigations are necessary to elucidate synergistic or additive effects of different substances in city smog and also the relative toxicity of each pollutant. Taking into account that more than 100 chemical sub* stances have been detected so far in air borne particulate matter, further analysis of chemical composition and biological effects are essential to evaluate potential risk of cytotoxicity for human beings as has been em- phasized by Sawicki [ 10].

Figs. 2 and 3. Loss of cell viability of mouse macrophages (line IC-21) in the presence of city smog extract and its fraction, estimated by dye exclusion test using pulse cyto- photometry after 5 and 24 h of incubation.

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REFERENCES

1 N. Seemayer, N. Manojlovic and A. Brockhaus, Zentralbl. Bakteriol., Parasitenkd., Infektionskr. Hyg., Abt. 1, Orig. B, 162 (1976) 27.

2 C.C. Schiirer, N. Manoj]ovic and N. Seemayer, Murat. Res., 74 (1980) 164. 3 N. Seemayer, Staub Reinhalt. Luft, 38 (1978) 254. 4 J. Mauel and V. Defendi, J. Exp. Med., 134 (1971) 335. 5 N. Secmayer, N. de Ruiter, N. Manojlovic and H. Weisz, Zentralbl Bakteriol., Para-

sitenkd., Infektionakr. Hyg., Abt. 1, Orig. B, 165 (1977) 260. 6 V.F. Garry and R.D. Moore, Oncology, 29 (1974) 429. 7 V.I. Oyama and H. Eagle, Prec. Soc. Exp. Biol. Med. (N.Y.), 91 (1956) 305. 8 W. Dehnen, N. Pitz and R. Tomingas, Cancer Letters, 4 (1977) 5. 9 A. Brockhaus, G. R~nicke and H. Weisz, Benzpyren-Pegel des Lufstaubs in der

Bundesrepublik Deutschland. Deutsche Forschungsgemeinachaft, Kommission zur Erforschung der Luftverunreinigung, Mitt. XIII, H. Boldt-Verlag, Boppard, 1975.

10 E. Sawicki, Chemical composition and potential "genotoxic" aspects of polluted atmospheres, in, U. Mohr, D. Schmi/hl and L. Tomatis (Eds.), Air Pollution and Cancer in Man, I.ARC Scientific Publication No. 16, International Agency for Re- search on Cancer, Lyon, 1977, pp. 127--187.

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