In vitro effects of fly ash on alveolar macrophages

Ctinsernation & RecycZing, Printed in Great Britain

Vo1.7,No.2-4,

pp.361-366,

1984

0361-3658184 Pergamon

IN VITRO --

EFFECTS

OF FLY ASH ON ALVEOLAR

$03.00/0 Press Ltd.

MACROPHAGES

W.K. Liu*, S.W. Tsao' and J.W.C. Wang** * Department of Anatomy, and ** Department of Biology The Chinese University of Hong Kong, Shatin, Hong Kong

ABSTRACT The cytotoxicity of fly ash particles on alveolar macrophages were studied using an -in vitro Rat alveolar macrophates were harvested from pulmonary lavage and incubated culture system. The fly ash particles used for investigation were from with fly ash particles for 24 h. The particle two sources: coal-fired power plant (FA I) and municipal incinerator (FA II). size distribution and the metallic contents (Cr, Cd, Cu, Fe, K, Mg, Mn, Ni, Pb and Zn) of The levels of several metallic elements (Fe, Mg and Cr) were the fly ash were measured. FA II particles, however, have considerably higher concentrations very high in both samples. FA II was found to be more (ten to hundred-fold) of Cd, Mn, Pb, Cu, K and Zn than FA I. toxic than FA I on cultured alveolar macrophages as indicated by the cell viability (trypan blue exclusion) and the amount of lactate dehydrogenase released into the culture medium. The difference in cytotoxicity of the two fly ash samples was confirmed under scanning electron microscope.

INTRODUCTION Fly ash is generated during combustion of coal in the coal-fired power plant furnace and solid waste in the municipal incinerator. They are fused solid residues coated with vaporised organic matters and inorganic metallic elements. Most of the fine particulates are carried in the effluent stream to the exhaust and are removed by electrostatic precipitation. a considerable amount will escape precipitation and is emitted into the atmosNevertheless, phere. Fly ash is regarded as an important air pollutant not only because of its small particle size, but also due to its enriched metallic content. Some of these metals, e.g. nickel, cadmium and lead have been reported to be toxic or carcinogenic[1,2]. chromium, These air-borne particles, once inhaled into the lungs, are phagocytosed by the alveolar a very important defence mechanism to protect the alveolar macrophages which constitutes region of the lungs. The object of this study is to use an -in vitro culture system of alveolar macrophages to evaluate the cytotoxicity of fly ash collected from two different sources in Hong Kong: (if coal-fired power plant, and (ii) municipal incinerator. Cytotoxicity was determined by the level of lactate dehydrogenase released into the culture medium and the morphological changes of alveolar macrophages co-cultured with fly ash particles as demonstrated by scanning electron microscopy.

MATERIALS

AND METHODS

FLY ASH SAMPLES Fly ash samples were obtained from two sources: the electrostatic precipitator of the smokestack of the coal-fired power plant (FA I) and that of the municipal incinerator (FA II). The samples were digested by mixed acidC31 and the contents of Cr, Cd, Cu, Fe, K, Mg, Mn, Ni, Pb and Zn in the fly ash samples were analysed with an atomic absorption spectrophotometer (Varian 1471).

361

W.K.

362

Liu, S.W. Tsao and J.W.C. Wong

The samples were hand ground in a mortar and heat sterilised at 180°C for 1 h before being resuspended in Dulbecco's modified Eagle's medium (DMEM). The suspension was ultrasonicated for 30 min. to disperse the particles in the medium before they were used for macrophage culture.

ALVEOLAR

MACROPHAGE

CULTURE

A female albino rat (about 150g body weight) was anaesthetised by intraperitoneal injection of 1.5 ml of 3.5% chlorohydrate. The lungs were dissected out and washed with 4 x 5ml of sterile phosphate buffered saline. The lavage fluid was pooled and centrifuged at 500g for 5 min. and the cell pellet was resuspended with DMEM to give a concentration of 1 x 10-6 cells ml-l. The alveolar macrophages thus harvested were allowed to attach onto the sterile glass cover slips in multi-well culture plate (Falcon) and incubated at 37-C in a humidified atmosphere containing 5% CO2 for 1 h. The non-adherent cells were removed by washing vigorously three times with culture medium. The culture prepared by this method contained more than 95% macrophages as observed by their staining characteristics, size and phagocytic activities. The viability rate of the cells was above 90% as demonstrated by trypan blue dye exclusion method[4]. Fresh culture medium containing different concentrations of fly ash (50, 250 and 500 ug ml-') was then added to the culture wells. The cultures were further incubated for 24 h and the lactate dehydrogenase released into the culture medium was determinedi51. The macrophages on the cover slip after culture were processed for scanning electron microscopy.

PREPARATION

OF ALVEOLAR

MACROPHAGES

FOR SCANNING

ELECTRON

MICROSCOPY

The alveolar macrophages attached to the glass slips were rinsed in phosphate buffer (pH 7.2) and fixed in 2.5% glutaraldehyde (in O.lM cacodylate buffer, pH 7.2). After fixation for lh, the cells were rinsed with O.lM cacodylate buffer and post-fixed with 1% osmium tetraThe cells were then dehydrated in graded concentrations of ethanol, immeroxide for 20 min. sed in Freon, critical point dried, coated with gold and observed in a JEOL 35CF scanning electron microscope.

STATISTICS The significance of the difference between the two treatment groups and the released from the cultured macrophages was determined using a 2-way analysis Further individual comparisons were done incorporating the Tukey TestC61.

LDH activity of variance.

RESULTS METALLIC

CONTENTS

IN FLY ASH

The contents of Cr, Cd, Cu, Fe, and shown in Table 1. Fe, K, and FA II. On the whole, the higher than that in FA I, except

LACTATE

DEHYDROGENASE

K, Mg, Mn, Ni, Pb and Zn in FA I and FA II were determined Mg and Zn were found to be particularly high in both Fa I metal content in FA II ranged from one to a thousand times the Fe content which is twice as high in FA I.

(LDH) ACTIVITY

LDH released into the culture medium showed a dose-dependent relationship with increasing Stimulation of PeleaSe of LDH is significantly concentration of fly ash I and II (Fig.1). higher in 3 different concentrations (50, 250 and 500 ug ml-l ) of FA II than those in FA I (p
Effects

Table

i.

of Fly Ash on Alveolar

Metal concentrations

(pg g

-1

363

Macrophages

) of two different fly ash Samples FA I/FA II

FA I

FA II

Fe

19570.99 + 595.63X

11440.66 _+ 2970.70

1:0.58

ME:

12470.40 k 665.50

16366.87 +

272.10

1:1.31

30.31

717.43 +

229.07

1:1.96

Cr

364.33 +

N1

106.08 ?:

3.28

170.18 +

18.95

1:1.60

Cd

35.03 +

1.43

205.76 +

23.49

1:5.87

Mn

323.17 +

4.77

3116.50 +

818.30

1:9.64

cu

48.53 +

3.14

1619.26 +

500.89

1:33.37

Pb

117.50 +

3.14

7314.86 i

2173.80

1:62.25

K

749.77 +

87.56

230254.60 + 40036.00

1:307.1

77.30 f

8.78

120801.29 + 33612.30

1:1562.7

Zn

*Mean ? Standard Deviation

Fig.1.

Lactate dehydrogenase activity in medium (24 h incubation). Vertical bars indicate the standard deviations of the means.

364

W.K. Liu, S.W. Tsao and J.W.C. Wong

MORPHOLOGICAL

OBSERVATION

The appearance of the macrophages after 24 h of culture was examined under a scanning electron microscope. The cells appeared to attach well to the surface of the culture dish and showed extensive undulations of cell membrane (Plates 1 and 2). The phagocytic activity was demonstrated by co-culturing the cells with heat-treated samples of Candida albican (Plates 3 and 4). When fly ash particles were added to the cultures and incubatedfor and the membrane of the h, the particles were seen to attach readily to the macrophages cells appeared to be perforating and disintegrating, suggesting cell damage (Plates 5 and The cytotoxic effects were also confirmed by Trypan blue staining which showed a high 6). proportion (usually above 75%) of non-viable cells. Finger-like cytoplasmic projections were also seen to extend from the macrophages to become entangled with the dust particles (Plate 7). Larger pieces of dust particles were occasionally partially engulfed by the macrophages (Plate 8). More extensive cell damage was found in culture with FA II than that with FA I and also when higher concentrations of fly ash were used (250 and 5OOug ml-').

DISCUSSION Alveolar macrophages are also called "dust cells" because of their ability to phagocytose particulate matter. They represent the first line of a cell defence mechanism against inhaled particles and therefore can be used in the evaluation of health hazards to the respiratory system of atmospheric fine particulates. It has been reported that particles coated with nickel, lead, manganese, cadmium, chromium, mercury, copper and iron can cause decreases in cell viability, phagocytic activity, oxygen consumption, glucose metabolism, cell membrane potential and lyzosomal enzymes in alveolar macrophages[7-111, Cellular damage was also observed when metal-coated particles came in contact with the plasma membrane or were enclosed inside the phagocytic vacuoles of the alveolar macrophages[l21. In both cases, there were disturbances of cell membrane and therefore cytoplasmic enzymes e.g. LDH was released from the damaged cells[12-141. In our study, a dose-dependent release of lactate dehydrogenase from the cultured macrophages, damage of plasma membrane and thereafter disintegration of the macrophages and a high percentage of dead cells stained by trypan blue exclusion were observed. The higher toxicity was found in the FA II which has a high metallic enrichment. These cytotoxic effects of the fly ash may be attributed partly to the high metallic contents found in both types of fly ash particles. such as reHowever, other materials, condensed hydrocarbons and other organic compounds, which are also present in the fly ash may also contribute to its cytotoxicity. The relative cytotoxicity of individual components is being investigated further. The aim of this study is to compare the cytotoxicity of various atmospheric particulates by using an -in vitro macrophage culture system. Results from incinerator fly ash and power plant coal ash show that different chemical properties of test substances can cause various degree of LDH release and cell damage. This assay system may therefore be applied to provide primary information for further -in viva investigations.

ACKNOWLEDGEMENTS The authors with to thank Prof. D.J. Riches for his valuable comments, Mrs H.J. Hou-Chan and Mr K.C. Cheung in the preparation and Miss J.S.K. Tam for their technical assistance of photographic plates and figures.

REFERENCES 1. 2. 3.

4.

Fisher, G.L. & Lammert, J.E., Mutagenicity of filtrates from respirable Crisp, C.E., coal fly ash. Sci. 199: 73-75, (1978). Fisher, G.L., chrisp, C.E. & Raabe, O.G., Physical factors affecting the mutagenicity of fly ash from a coal-fired power plant. G. 204: 879-881, (1979). Allen, S.C., Grimshaw. H.M., Parkinson, J.A. & Quarmby, C. Chemical Analysis of Ecological materials. Oxford, Blackwell (1974). In: Tissue Culture Methods Dye exclusion tests for cell viability. Phillips, H.J., New York, Academic Press (1973) and Applications. (Eds Kruse, P.R. & Putterson, M.K.).

Effects

Plate

of Fly Ash on Alveolar

1:

Control

2:

Control alveolar macrophages is spherical in shape and attached Extensive surface undulation membrane is the to the surface. characteristic of alveolar macrophage.

3/4: Alveolar

alveolar

macrophages

365

Macrophages

macrophage

after culture

with engulfed

Candida ~~

for 24 h.

albican

5/h: Alveolar macrophage with fly ash particles. surface undulation membrane is lost. 7:

Tangled cytoplasmic ash particle.

processes

8:

Alveolar

with engulfed

macrophage

of alveolar

Characteristic

macrophage

fly ash particle.

on the fly

366

5. 6. 7.

8. 9.

10.

11.

12.

13. 14.

W.K. Liu, S.W Tsao and J.W.C.

Wong

Wroglewski, F., & La Due, J.S., Lactic dehydrogenase activity in blood. Pr0c. sot. Biol. Med. 90: 210-213, (1955). Weber, E., Grundriss der Biologischen Statistic. 7 Aufl. Stuttgart Aranyi, C.F., Miller, J., Andres, S., Ehrlich, R., Fenters: ~~7*'dardner, D.E. & Waters, M.E., Cytotoxicity to alveolar macrophages of trace metals absorbed on fly ash. Environ. Res. 20: 14-23, (1979). Camner, P., Johansson, A. & Lundborg, M., Alveolar macrophages in rabbits exposed to nickel dust. Environ. Res. 16: 226-235, (1978). Castranova, V., Bowman, L., Reasor, M.J. & Miles, P.R., Effects of heavy metal ions on selected oxidative metabolic processes in rat alveolar macrophages. Toxicol. APP~. Pharmacol. 53: 14-23, (1980). Garrett, N.E., Campbell, J.A., & Stack, H.F., The utilisation of the rabbit alveolar macrophage and Chinese hamster ovary cell for evaluation of the toxicity of particulate materials. Environ. Res. 24: 345-365, (1981). Graham, J.A., Gardner, E., Waters, M.D. & Coffin, D.L., Effects of trace metals on phagocytosis by alveolar macrophages. Infect. & Immun. 11: 78-1283, (1975). Gormley, I.P., Wright, M.O. & Ottery, J., The effects of toxic particles on the electrophysiology of macrophage membranes. Ann. Occup. Hyg. 21: 141-149, (1978). Adamis, Z. L Timar, M., Studies on the effect of quartz. bentonite and coal dust mixtures on macrophages -in vitro. Brit. J. Exp. Pathol. -12: 2;7-236, (1978). Beck, E.C., Interaction between fibrous dust and cells in vitro. Ann. Anat. Pathol. 12: 227-236, (1976).