In vitro cytotoxicity studies of particulate samples in cultures of Hamster Tracheal Epithelial (HTE) cells

Pergamon

0887-2333(94)00116-2

Toxic. in Vitro Vol. 8, No. 4. pp. 735-738, 1994 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0887-2333/94$7.00+ 0.00

IN VITRO CYTOTOXICITY STUDIES OF PARTICULATE

SAMPLES IN CULTURES OF HAMSTER TRACHEAL EPITHELIAL (HTE) CELLS A. KUPER-SMITH,J. N. LAWRENCEand D. J. BENFORD Division of Chemical Safety Evaluation, Robens Institute of Health and Safety, University of Surrey, Guildford, Surrey GU2 5XH, UK Abstract--Airborne contaminants have been implicated as important contributing factors in the aetiology of tumours of the respiratory tract. A major concern is that the potential carcinogenic activity of the particulate matter may be enhanced by the volatile material, which is adsorbed onto the surface. The cytotoxic potential of complex particulates was evaluated using the Neutral Red uptake method in primary cultures of hamster tracheal epithelial cells. Cytotoxicity of the reference particulates (benzo[a]pyrene adsorbed on to the surface of alumina A, alumina B, ferric oxide and titanium dioxide) was increased when cultures were treated with adsorbed rather than desorbed (plus dimethyl sulfoxide)samples. Foundry fume samples did not show the same effect.

XIV) from Boehringer Mannheim (Lewes, UK) and benzo[a]pyrene (BP; 99%) from Aldrich (Gillingham, Dorset, UK). Particulates (size < 8.5 #m) used were alumina A ('Specpure' alumina) and ferric oxide (iron [Ill]oxide) (supplied by Johnson Matthey Chemicals Ltd, Royston, Herts., UK), alumina B ('Spherisorb' 5 p m alumina; Phase Sep. Ltd, Queensferry, Clwyd, UK) and titanium dioxide (AR grade, BDH, Dagenham, Essex, UK). Foundry fume samples (mean median diameter < 5 gm), namely greensand with coal dust binder, greensand with coal dust substitute, Ashland process and shell moulding process (reference numbers: CW/80716, RL/81166, CW/80714 and CW/80715, respectively) were provided by Dr P. A. Ellwood, Health and Safety Executive.

INTRODUCTION

Cancer of the respiratory tract accounts for 14% of total cancer deaths in women and 34% in men (Mass and Kaufman, 1984). Cancer of the bronchial epithelium is the most common (Cohen and Moore, 1976), primarily resulting from the inhalation of tobacco smoke. Airborne contaminants have been implicated as important contributory factors in the aetiology of tumours of the respiratory tract. A major concern is that the potential carcinogenic activity of the particulate matter may be enhanced by the volatile material, which is adsorbed onto the surface. The work described here evaluates the cytotoxic potential of complex particulates using the Neutral Red uptake method in primary cultures of hamster tracheal epithelial cells (HTE). Test materials include foundry fume samples, reference particulates and benzo[a]pyrene adsorbed onto the surface of particulates.

Methods

MATERIALS AND METHODS

Materials

Golden Syrian hamsters (Wrights, Essex, UK), 5-10 wk old, were kept in cages at 22 ___2°C and a relative humidity of 55-65% using a 12-hr light/dark cycle. Food and water were supplied ad lib. All chemicals were purchased from Sigma (Poole, Dorset, UK) unless stated otherwise. Culture medium, antibiotics and tissue culture plastic were obtained from Gibco (Paisley, UK), pronase (type

Abbreviations: BP = benzo[a]pyrene; DMSO = dimethyl sulfoxide; HTE cells = hamster tracheal epithelial cells.

Cell isolation and culture. Hamster tracheal epithelial (HTE) cells were isolated by cannulation of the trachea and overnight enzyme digestion using 1% pronase (Kuper and Benford, 1991). Cells were harvested by centrifugation (500g, 5 min) and subsequently cultured on collagen-coated 96-well plates at a density of about 4 x 104 cells per well, in medium consisting of Dulbecco's minimal essential medium: Ham's F12 (mixture 3:1) supplemented with 0.4/~g hydrocortisone/ml, 1 nM transferrin/triiodo-lthreonine, 18 mM adenine, 5 #g insulin/ml and 10 n u cholera toxin (Hoh et al., 1987). Sample preparation and cytotoxicity assay. BPcoated particulates (Kuper-Smith et al., 1993) and foundry fume samples were treated either by desorbing solvent-soluble organics from their surfaces with dimethyl sulfoxide (DMSO) prior to exposure of cell cultures, or direct addition of samples to the

735

A. KUPER-SMITH et al.

736

culture medium. Samples were vortexed vigorously, diluted in culture medium, so that desorbed samples included 1% DMSO and cells were treated immediately. Plates were left for 24 hr, with foundry fume samples/coated particulates allowed to settle on the cell surface. After 24 hr the cell layers were washed carefully with phosphate buffered saline, in order to remove as much of the sample as possible before performing the Neutral Red assay. Cytotoxicity assay. Neutral Red uptake was determined as described by Borenfreund and Puerner (1985). The destain solution added to the wells was transferred into a new 96well plate for spectrophotometric measurement of samples, as some of the particulate sample could not be removed from the cell surfaces and residues interfere with the measurement of optical density. Absorbance was read at 540 nm using an EL 340 microplate reader (Biotek Instruments, Southampton, UK) and results expressed as percentage of controls. Student’s t-test was used to analyse differences between control and test groups.

RESULTS

Results are presented in Table 1.

Table 1. Cytotoxicity of particulate samples on primary cultures of hamster tracheal epithelial ceils evaluated with the Neutral Red uptake assay Percentage of control It SD Treatment Alumina

I

Alumina

A&orbed particulates

A trend of increasing cytotoxicity was observed with increasing concentrations of BP adsorbed to particulates. This increase occurred at the top doses used for same particulate concentrations and seems to reflect increasing ‘BP-loading’. Desorption of BP from the surface of the particulate sample led to decreased toxicity compared with adsorbed samples and no correlation to increasing BP concentrations could be found (Fig. 1). Foundry fme

samples

No apparent difference was found in the cytotoxic

response of HTE cells to foundry fume samples, when exposed to adsorbed samples or samples, which had solvent extractable organics desorbed off the surface of the foundry fumes. Samples 66 and 15, namely greensand with coal dust substitute and she11moulding process, appeared to be cytotoxic at concen-

0

B

I 10 100 0

Ferric oxide

Titanium

dioxide

1

Particulates onlv 100 65.6 60.1 20.7

+ + + +

Particulates DlUS

21.9 14.7 11.9 15.2

lOOk21.7 71.4k20.2 58.6 It 23.8 29.4 i 17.3

100 It 114.71 56.7 f 4.5 f

100 * 15.9

99.5+ 16.0

10 loo

90.4+ 15.1 71.9 + 20.0

0 1 IO 100

100 72.3 59.6 53.8

Benzo[a]pyrene (dissolved in DMSO)

+ 16.3 If: 26.2 + 30.2 f 7.5

100 98.6 89.2 77.8 83.8

1

(n0.f 14

Percentage

0 0.01 0.1

1 IO 66

0 0.01 0.1 1 10 0 0.001 0.01 0.1

1 16

0 0.01 0.1 1 10

100 + 8.7 74. I + 4.7* 33.5 f 3.5** 7.7 * 4.S”

Percentage of control + SD

0 0.1

DOSe (w/m0

7.7 11.8 1o.v 0.1++*

loot_ 7.3 83.5 k 19.8 62.5 _I 20.3 33.7*4.1**

10 loo Foundary fume samples

DMSOt

100+8.3 96 f 5.8 10.8 + 8.S’ 2.8 + IX?**

Dose (a@

15

No cytotoxicity was observed with BP at concentrations up to 100pM.

0

A

10 100

Particulates alone

A dose response was observed with increasing concentrations of particulates. Addition of DMSO to the culture system led to a slight increase in cytotoxicity, when compared with cultures treated with the particulate alone.

DOW (maImit

of control I: SD

Adsorbed 100 93.8 97.1 110.5 88.3

* f f + +

f 19.8 + 8.0 f- 17.1 I21.6 i 26.4

17.4 14.7 36.6 12.7 22.2

Desorbed 1OOi 15.6 118.6 + 20.3 120.6 f 19.7 117.3+21.8 28.0 + 2.5*“*

IO0 99.3 83.2 38.4 38.5

+ 18.2 + 19.4 + 17.4 &. l7.6** + 25.7+*

100 94.4 79.4 27.6 17.7

f 15.7 + 20.0 +_ 14.4 f 8.6”” + 3.1***

100 119.1 97.8 87.9 17.9

+ 15.5 + 19.9 & 23.0 + 14.7 _t g.7***

100 102 92.2 92.7 19.9

i. rt f f *

100 + 106.2 + 114.3 * 121.2 + 42.6 +

16.1 13.8 10.1 29.5 20.3”

100 f 93.6 + 99.7 k 112.4 + 39.8 +

17.7 29.9 30.4 16.8 1I .7**+ 14.3 3.4 18.4 24.6 21.4**

DMSO = dimethyl sylfoxide Cultures were treated with the test compounds for 24hr. Results are expressed as percentage control + SD for three or more experiments with three or more replicates per dose group. )n = two experiments with three or more replicates. Values with superscript differ signi~~ntly from control (Student’s t-test: ‘P < 0.05; **p < 0.01; **+p < 0.001).

trations of 1 mg/ml, whereas the test compounds no. 14 and 16, namely Ashland process and greensand with coal dust binder, showed a cytotoxic response at the highest tested concentrations. No differences were seen in cytotoxicity with adsorbed or desorbed samples, except for sample 14, which was more toxic following desorption at the top concentration tested.

Cytotoxicity of particulates in HTE cells 160

16o [

737

[

140 120

1I 1I2-0.1-0' 0~1t l i i4~ ...................................................... 0 ii~iiiiiiiiii_~!!

!

i

:

100 80

60 ......................... I ..........

. . . . . . . . .

60 40 20 0

20~

..............................................................................

(a/ I

I

I

I

I

I

I

I

0.0060.0140.0550.060.141

I

I

I

0.55 01.6 1.4

I

0.008 0.021

0.08 0.21

0.8

2.1

on 0.038 mg particulate

on 0.38.mg particulate

on 3.8 rna particulate

515

nl

(b) i

I

i

I

I

I

on 0.0!6 mg partieul~tte

160

on 1.6 ma particulate

::iii iiiiiiii iiiiiiii iii

120 ............................................................................................

80

0

I

140 .....................................................................................................

100

20

I

160,

120

4O

i

Dose (p.M BP)

a. 140

60

i

on .0,,1~.mg pameumte

Dose (p.MBP) o

I

0.006 0.019 0.051 0.06 0.19 0J51 01.6 ' 1.9 51.1 0.009 0.023 0.09 0.23 0.9 2.3

iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii iiii 20i ........................................................................................................

(c) I

(d)

I

I

I

I

I

1

I

I

I

I

I

0.008 0.037 0.051 0.08 0.37 0.~11 0]8 ' 317 I 5.1 0.01 0.043 0.1 0.43 1 4.3 on 0.08.2mg particulate

on 0.82.mg particumte

on 8.2 m¢l particulafe

Dose (p.MBP)

0 ~ IIII

I

I

0.012 o.032 0.~5 032 0.;2 o.o17o.o37

I

0[5

o17 037

on 0.045 rng

on 0.45.mg

particulate

particulate

11111 1.2

17

3.2

3z

;

on 4.5 m~ particulate

Dose(ttM BP)

Fig. 1. Cytotoxicity of benzo[a]pyrene (BP)-coated particulates (a, alumina A/BP; b, alumina B/BP; c, ferric oxide/BP; d, titanium dioxide/BP) on primary cultures of hamster tracheal epithelial cells measured with the Neutral Red assay. Cultures were treated for 24 hr with BP either adsorbed onto the particulate surface (D) or desorbed from their surface with dimethyl sulfoxide (I--I). Results are presented as mean __+SD for three or more experiments,triplicate cultures per dose group. *Test data differ significantly from control (Student's t-test: P < 0.05).

DISCUSSION A synergistic effect of particulate/BP samples was observed with concentrations of BP and particulate that had previously shown no effect, either individually or as mixtures together (not adsorbed) within the limited range of concentrations of BP adsorbed onto the surface of particulate samples. No cytotoxicity was observed after administration of BP desorbed from the surface of the particulate sample. Cell viability was reduced at the highest concentrations of BP adsorbed onto particulates to 40% of control levels with alumina A/BP, alumina B/BP and ferric oxide/BP, and to about 60% with titanium dioxide/BP. Although it is not clear why the addition of DMSO to the testing of ferric oxide particles alone should increase its cytotoxicity especially with concentrations of 10mg/ml or above, it is possibly attributable to the addition of DMSO leading to a membrane effect and thereby increasing toxicity.

Adsorption of BP to the surface of particulates may enhance the transfer of BP into membranes, as compared with uptake from the microcrystalline states. It is also possible that the localized higher concentration of BP when adsorbed onto the surfaces of particulates is responsible for the increased cytotoxicity. It has been shown that the availability and metabolism is greater for chrysotile, ferric oxide or silicon as carrier, than for carbon black or diesel exhaust (Bevan and Manger, 1985; Leung et al., 1988). The data presented for foundry fumes imply that the cytotoxic effect is due to the particulate matter rather than any adsorbed organics, such as polycyclic aromatic hydrocarbons, with the only increase in cytotoxicity observed with sample 14. The increase observed was opposite to that expected, if a localized concentration of adsorbed matter was responsible for the eytotoxic effect. It can only be speculated whether this effect was due to enhanced cytotoxicity of the particles in the presence of DMSO, or to the desorption of previously biologically inactive DM SO-soluble organics from the particulate surface.

A. KUPER-SMITH et al.

738

Acknowledgement--The authors gratefully acknowledge financial sponsorship from the Health and Safety Executive.

REFERENCES

Bevan D. R. and Manger W. E. (1985) Effect of particulates on metabolism and mutagenicity of benzo(a)pyrene. Chemico Biological Interactions 56, 13 28. Borenfreund E. and Puerner J. A. (1985) Toxicity determined in vitro by morphological alterations and Neutral Red adsorption. Toxicological Letters 24, 119 124. Cohen G. M. and Moore B. P. (1976) Metabolism of [3H]benzo(a)pyrene by different portions of the respiratory tract. Biochemical Pharmacology 25, 1623-1629. Hoh A.~ Maier K. and Dreher R. M. (1987) Multilayered

keratinocyte culture used for & vitro toxicology. Molecular Toxicology 1, 537 546. Kuper A. and Benford D. J. (1991) Unscheduled DNA synthesis in tracheal epithelial cell cultures. Toxicology in Vitro 5, 511-513. Kuper-Smith A., Lawrence J. N. and Benford D. J. (1993) In vitro effects of benzo(a)pyrene-coated particulates on hamster tracheal epithelial cells. Human and Experimental Toxicology 12, 425-426. Leung H. W., Henderson R. F., Bond J. A., Mauderley J. L. and McClellan R. O. (1988) Studies on the ability of rat lung and liver microsomes to facilitate transfer and metabolism of B(a)P from diesel particles. Toxicology 51, 1-9. Mass M. J. and Kaufman D. G. (1984) Biochemical studies of the tracheobronchial epithelium. Environmental Health Perspectives 56, 61-74.