Ann
-
OCCU
P- Hyg> V o L 4 1 - Supplement 1, pp. 32-38, 1997
( g 1 9 9 7 British Occupational Hygiene Society
Published by Elsevier Science Ltd. Alj rights reserved
Printed in Great Britain
0003^*878/97 $17.00 + 0.00
Inhaled Particles VIII
PII: S0003-4878(96)00130-5
SURFACE FREE RADICAL ACTIVITY OF PM10 AND
ULTRAFINE TITANIUM DIOXIDE: A UNIFYING FACTOR
IN THEIR TOXICITY?
P. Gilmour*, D. M. Brown*, P. H. Beswick*, E. Benton*, W. MacNeef
and K. Donaldson,
*Biomedicine Group, Department of Biological Science, Napier University, 10 Colinton Rd, Edinburgh
EH10 5DT, U.K.; and tUnit of Respiratory Medicine, University of Edinburgh, Edinburgh, U.K.
INTRODUCTION
Epidemiological evidence has been accumulating showing a strong relationship
between particulate environmental air pollution (PM10) and end-points of respiratory ill health such as attacks of asthma, COPD, diminished lung function and
cardio-vascular deaths (Pope et al., 1995). To date there has been no plausible
biological hypothesis to explain this relationship at the very low airborne mass
concentrations of particulate air pollution that are found (< 50 \ig ml" 1 ). We
recently hypothesised (Seaton et al., 1995) that an ultrafine (< 100 nm diameter)
component of PM10 is responsible for its adverse effects. This is based on the initial
studies of Oberdorster and colleagues (Feirin et al., 1992) who demonstrated that
titanium dioxide in the ultrafine form (20 nm diameter) was highly inflammogenic
to the lungs of rats compared to fine (200 nm diameter) TiO2 particles at the same
airborne mass concentration.
We now hypothesise that the adverse effects of PM10 on the lung result from free
radical activity at the surface of an ultrafine fraction. We further hypothesise that
the interstitialisation that was seen with UFTiO2 (Ferin et al., 1992) could similarly
occur with the ultrafine component of PM10. If the ultrafine material has free
radical activity then the increased surface area that is presented to the epithelial
surface by a relatively small mass of ultrafine particles could compromise epithelial
integrity leading to interstitialisation.
We demonstrate here that ultrafine TiO2 and PM10 both have hydroxyl radical
activity and that UFTiO2 is capable of causing hydroxyl radical-mediated membrane damage to erythrocytes; fine TiO2 has much less of these properties.
Additionally PM10 hydroxyl radical activity is either in the ultrafine fraction or is
released in soluble form.
MATERIALS AND METHODS
Particles
PM10 was collected from the Edinburgh sampling site of the enhanced urban
network and removed from the filters by sonication; a blank filter, similarly
32
Surface free radical activity of PM 10
Control
PM10 Mannitol
33
PM10 +
Mannitol
Fig. 1. Image of an agarose gel showing depletion of the supercoiled DNA band by PM 10 and protection
by mannitol.
treated, was used to control for any free radical activity released from the filters
themselves. Fine TiO2 was obtained from Tioxide Ltd with average particle
diameter of 583 nm and UFTiO2 from Degussa (P25) with average particle
diameter of 96 nm.
Plasmid assay for hydroxyl radical activity
The ability to break strands of supercoiled plasmid DNA was the sensitive assay
used here for detecting particle surface-associated free radicals (Gilmour et al.,
1995). Briefly qp XI74 RF plasmid DNA was incubated with the differing quantities
of particles. All treatments were carried out in a volume of 20 ul, achieved by the
addition of filtered ultra-pure distilled water. Each treatment or control sample was
incubated at 37°C in a water bath for 8 h. The three plasmid forms (super-coiled,
relaxed coil and linearised plasmid) were separated by electrophoresis for 16 h at
30 V on a 0.8% agarose gel. The proportion of the plasmid forms, which provide a
measure of the free radical damage to the plasmid, were quantified by scanning
laser densitometry and free radical damage to DNA was expressed as depletion of
the supercoiled DNA band. To confirm the role of hydroxyl radicals in the DNA
damage, mannitol was used as a scavenger.
Damage to the plasma membrane of sheep erythrocytes
As a model membrane target, we utilised the sheep erythrocyte membrane, and
ascertained the ability of TiO2 samples to cause direct membrane damage. Washed
sheep erythrocytes were uncubated for 30 min with differing concentrations of
particles and membrane damage was assessed as the absorbance at 540 nm
(haemoglobin) in supernatants of centrifuged samples. Again, mannitol was used
to confirm the role of hydroxyl radicals and DSF-B was used to investigate the role
of iron.
RESULTS
PM10
PM10 had free radical activity that caused scission depletion of supercoiled
plasmid DNA as shown in Fig. 1. This could be protected against with mannitol,
clearly implicating hydroxyl radical in the injury.
We quantified the amount of DNA in the supercoiled ban using scanner laser
densitometry and image density analysis software (Fig. 2) revealing that a blank
filter yielded very little free radical activity whilst the PM10 loaded filter gave up to
34
P. Gilmour et al.
ile
Q
•a
o
<5
a3
(0
"5
leti
o
a.
100
90
80
70
60
50
40
30
20
10
T
T
01
Filter
PM10 Mannitol PM10+
Man
DSF-B
PM10 +
DSF-B
Treatment
Fig. 2. Free radical activity of PM10 material and amelioration by mannitol (Man) and by
desferoxamine-B (DSF-B); results are mean ± sem of at least three separate experiments.
90% depletion of the supercoiled band. Confirmation of the role of hydroxyl
radical and iron were shown by the blockage of the free radical injury by mannitol
and desferoxamine-B, respectively, in Fig. 2.
Evidence that the free radical activity emanates from an ultrafine fraction comes
from studies where the PM10 was centrifuged to clarity and was found to have the
same free radical activity as the standard PM10 material (Fig. 3). Another
interpretation of this result is of course that the PM10 releases a soluble component
that is responsible for the hydroxyl radical; this involves iron as evidenced by the
inhibition with DSF-B shown in Fig. 3.
100.0
90.0
t
80.0 4
|
70.0 4
60.0 -[•
« 50.0 i
0 40.0 {
I
1 30.0 -f20
f^ 10.0° I4
o.o 4PM10
Cent
PM10
DSFB
Cent
PM10
DSF-B
Treatment
Fig. 3. Free radical activity, as shown in the plasmid assay, of PM10 material and PM10 material
centrifuged to clarity (Cent PM10) and the blocking effect of Desferoxamine-B (DSF-B).
Surface free radical activity of PM10
I
Ultrafine
TiO2
35
TiO2 ng/ml
Fig. 4. Image of a supercoiled DNA band in an agarose gel to show depletion of the supercoiled DNA
band by UFTiO2 and relative lack of free radical activity of TiO2.
Ultrafine TiO2
When UFTiO2 and TiO2 were studied in the plasmid assay for hydroxyl radical
activity the UFTiO2 was found to have substantially more activity than TiO2 (Figs.
4 and 5). As an alternative assay for the free radical-injuring activities of UFTiO2
and TiO2 we measured the abilities of the two samples to cause haemolysis of sheep
erythrocytes. As shown in Fig. 6, only UFTiO2, not TiO2, was able to cause
haemolysis in a concentration dependent manner. Figure 7 shows that the
haemolytic effect of UFTiO2 was blockable with mannitol at higher concentrations,
confirming the role of hydroxyl radical in the haemolysis caused by UFTiO2.
Iron involvement in the haemolytic effect of UFTiO2 could not be demonstrated
using DSF-B, but we cannot yet discount interference from haemoglobin as a
confounding factor.
DISCUSSION
We demonstrate here that PMi0 material and UFTiO2 both have hydroxyl
radical activity that is detectable in a supercoiled plasmid DNA assay and that
UFTiO2 also has the ability to cause hydroxyl radical-mediated haemolysis of sheep
erythrocytes. Unfortunately, because of the small amounts of PM10 currently
so
Concentration
100
150
(ug/ml)
Fig. 5. Depletion of supercoiled DNA by UFTiO2 and TiO2 as quantified by scanning laser densitometry
of the supercoiled bands.
36
P. Gilmour et al.
2
4
6
8
10
Concentration (mg/ml)
Fig. 6. Haemolytic activity of UFTiO2 and TiO2: results are mean ± sem of three experiments.
available we have been unable to test the haemolytic activity of PM10. TiO2 has
barely detectable free radical activity in the plasmid assay and is unable to cause
haemolysis at equivalent mass to UFTiO2. Quartz has been reported to cause
haemolysis by an oxidative mechanism, blockable with catalase (Razzaboni and
Bolsaitis, 1990), but catalase had no effect on the haemolysis caused by UFTiO2
here (data not shown).
The particle size of the PM10 material used here is unknown but the size sampling
characteristics of the tapered element oscillating microbalance that is used in the
Edinburgh sampling site of the enhanced urban network follows the PM10
convention and collects 10 u.m aerodynamic diameter particles with 50% efficiency.
0
5
1
0.5
0.1
0.05
0.01
Mannitol (mM)
Fig. 7. Amelioration of UFTiO2-mediated haemolysis with mannitol; mean ± sem of four experiments.
Surface free radical activity of PMj
30
25
(0
>t
20
37
UFTiO2
X
T
1
X
1
2
4
o
15
E
0)
CO
X
10
0
0.5
DSF-B (mM)
Fig. 8. Effect of iron chelation with DSF-B on UFTiO2-mediated haemolysis.
It also, however, collects a smaller proportion, by number, of larger particles and
collects particles down to the ultrafine range, very likely in large numbers (Quality
of Urban Air Review Group, 1996). We have no information on the harvesting
efficiency of our method for retrieving PMi0 from the filters for different particle
sizes. However, we believe that there is a large proportion, in particle number and
surface area terms, of ultrafine particles in the PM10 used in these experiments. We
also hypothesise that the hydroxyl radical activity resides in the ultrafine fraction of
the PM10. This was supported by the fact that in a sample of clear supernatant from
which PMio particles had been removed by centrifugation, there was the same
amount of free radical activity as in the whole suspension. However, an alternative
explanation is that the "hydroxyl generating system" which involves iron, as shown
by DSF-B studies, diffuses from the PM10 particles into solution.
The marked difference in hydroxyl radical activity of UFTiO2 and TiO2 could
underlie the difference in pathogenicity found with these two materials by Ferin et
al. (1992) and the differential expression of anti-oxidant genes in the lungs of rats
inhaling UFTiO2 or TiO2 (Janssen et al., 1994).
The hydroxyl radical-mediated haemolysis caused by UFTiO2 could not be
inhibited with DSF-B, ruling out Fenton chemistry as the source of the hydroxyl
radical for this particle but this could be a result of free iron from haemoglobin
binding to desferrioxamine and confounding the assay. In preliminary studies
however, using the plasmid assay, there was no protective effect of chelation with
desferoxamine on the free radical damage caused by UFTiO2.
The involvement of iron in the hydroxyl radical-generating activity of PM10 but
not in the case of UFTiO2, if this is confirmed, is a clear difference between the two
paniculate materials. UFTiO2 had an average particle diameter of 96 nm and TiO2
had an average particle diameter of 583 nm. Particle surface area calculations
indicate that the maximum difference in surface area is 7 times more surface area of
UFTiO2 compared to an equal mass of TiO2. Consideration of the data given above
38
P. Gilmour et al.
show that the differences in the hydroxyl radical activity between TiO2 and UFTiO2
cannot be explained by differences in surface area because in excess of a sevenfold
increase in TiO2 still did not cause the same degree of plasmid-damaging or
haemolytic activity as UFTiO2.
In conclusion, the data shown here reveal that both PM10 and ultrafine TiO2
have hydroxyl radical-generating activity. Preliminary studies with UFTiO2 suggests that the source of the free radical activity is different for these two particles,
involving iron in the case of PM10 but not in the case of UFTiO2. However, the
latter finding could be artefact due to the presence of haemoglobin in the
haemolysis assay.
The data do not establish that the hydroxyl radicals emanate from an ultrafine
component in the PM10 but generally support this contention. An alternative
explanation is that the hydroxyl radical generating system, which involves iron,
diffuses into solution from the PM10 particles. The latter would be likely to occur in
the lung and the modifying effect of components of the lung lining fluid on this
process are under investigation.
Acknowledgements—We acknowledge the financial assistance of the Colt Foundation and the British
Occupational Hygiene Research Foundation.
REFERENCES
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in rats. Am. J. respir. Cell Mol. Biol. 6, 535-542.
Gilmour, P., Beswick, H. P. and Donaldson, K. (1995) Surface free radical activity of a range of
respirable industrial fibres assessed using (j>x 174 RF1 plasmid DNA. Carcinogenesis 16, 2973-2979.
Janssen, Y. M., Marsh, J. P., Driscoll, K. E., Borm, P. J., Oberdorster, G. and Mossman, B. T.
(1994) Increased expression of manganese-containing superoxide dismutase in rat lungs after
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for reassessment?. Environ. Health Perspect. 103, 472-480.
Quality of Urban Air Review Group (1996) Airborne particulate matter in the United Kingdom, 3rd
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Razzaboni, B. L. and Bolsaitis, P. (1990) Evidence of an oxidative mechanism for the hemolytic activity
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Seaton, A., MacNee, W., Donaldson, K. and Godden, D. (1995) Particulate air pollution and acute
health effects. Lancet 345, 176-178.