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Guumlunter Oberduumlrster studies

in vivoToxicology of ultrafine particles

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101098rsta20000680

Toxicology of ultramacrne particles in vivo studies

By G unter Oberd orster

Department of Environmental Medicine School of Medicine and DentistryUniversity of Rochester Rochester NY 14642 USA

Ultrashy ne particles (less than 100 nm in diameter) are encountered in ambient air andat the workplace Normal background levels in the urban atmosphere for ultrashy neparticles are in the range 14 pound 104 cmiexcl3 however their mass concentration is nor-mally not greater than 2 m g miexcl3 At the workplace ultrashy ne particles occur regularlyin metal fumes and polymer fumes both of which can induce acute inregammatoryresponses in the lung upon inhalation Although ultrashy ne particles occurring at theworkplace are not representative and therefore are not relevant for urban atmo-spheric particles their use in toxicological studies can give valuable information onprinciples of the toxicity of ultrashy ne particles Studies in rats using ultrashy ne polymerfumes of polytetrareguoroethylene (PTFE) (count median diameter ca 18 nm) showedthat (i) they induced very high pulmonary toxicity and lethality in rats after 15 minof inhalation at 50 m g miexcl3 (ii) ageing of PTFE fumes resulted in agglomeration tolarger particles and loss of toxicity (iii) repeated pre-exposure for very short periodsprotected against the toxic and lethal enotects of a subsequent 15 min exposure (iv)rapid translocation of PTFE particles occurred to epithelial interstitial and endothe-lial sites Since one characteristic of urban ultrashy ne particles is their carbonaceousnature exposure of rats to laboratory-generated ultrashy ne carbonaceous (elementaland organic carbon) particles was carried out at a concentration of ca 100 m g miexcl3

for 6 h Modulating factors of responses were prior low-dose inhalation of endotoxinin order to mimic early respiratory tract infections old age (22-month old rats ver-sus 10-week old rats) and ozone co-exposure Analysis of results showed that (i)ultrashy ne carbon particles can induce slight inregammatory responses (ii) LPS prim-ing and ozone co-exposure increase the responses to ultrashy ne carbon (iii) the agedlung is at increased risk for ultrashy ne particle-induced oxidative stress Other studieswith ultrashy ne and shy ne TiO2 showed that the same mass dose of ultrashy ne particleshas a signishy cantly greater inregammatory potential than shy ne particles The increasedsurface area of ultrashy ne particles is apparently a most important determinant fortheir greater biological activity In addition the propensity of ultrashy ne particles totranslocate may result in systemic distribution to extrapulmonary tissues

Keywords ultramacrne particles carbon pulmonary indeg ammationdosimetry adaptation LPS age

1 Introduction

Epidemiological studies have shown an association between increased particulate airpollution and adverse health enotects in susceptible parts of the population in par-ticular the elderly with respiratory and cardiovascular diseases (EPA 1996) Urban

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2720 G Oberdorster

airborne particles consist of three modes ultrashy ne particles accumulation-mode par-ticles (which together with the ultrashy nes form the shy ne particle mode) and coarse-mode particles Ultrashy ne particles (particles less than 01 m m in diameter) contributevery little to the overall mass but they contribute most to the number concentra-tion of airborne urban particles Any of the three modes could causally be associatedwith adverse health enotects although epidemiological studies suggest that shy ne-modeparticles are better correlated with such enotects than coarse particles (EPA 1996) Weare testing the hypothesis that urban ultrashy ne particles consisting of a carbonaceouscore with attached inorganic and organic materials can cause adverse health enotectsin compromised subjects during episodic high increases in concentration

Ultrashy ne particles are also encountered in the workplace as fumes generated fromsmelting processes of metals and heating of polymers Resulting acute enotects inexposed workers have been well documented as metal-fume fever and polymer-fumefever consisting of acute pulmonary and inregammatory responses which can alsobe accompanied by systemic enotects with symptoms of fever nausea and headaches(Drinker et al 1927 Gordon et al 1992 Goldstein et al 1987 Rosenstock amp Cullen1986) Concentrations of such ultrashy ne particles at the workplace are very highcompared with those in the urban atmosphere and often the high concentrationsencountered at the workplace result in particles which have agglomerated by classi-cal coagulation phenomena to larger sized particles

Although workplace exposures to ultrashy ne fumes are known to result in poten-tially severe acute responses a direct extrapolation to enotects of urban ultrashy neparticles is not possible The toxicity of carbonaceous urban ultrashy ne particles isapparently much lower compared with that of freshly generated metal or polymerfumes However an important dinoterence between exposures to ultrashy ne particles atthe workplace and in the general environment lies in the exposed population Theseare healthy adults at the workplace and all of the population including the mostsensitive members in the urban setting Despite this it is likely that certain prin-ciples and concepts of ultrashy ne particle toxicology are common between workplaceultrashy ne particles and urban ultrashy ne particles Therefore it could be very useful toassess the toxicity and behaviour of ultrashy ne particles encountered at the workplacewhich might be helpful for designing studies with ambient ultrashy ne particles Thispaper will shy rst summarize some of our studies with polymer fumes discuss certainprinciples of ultrashy ne particle dosimetry and behaviour and shy nally describe resultsof toxicological studies with carbonaceous and other ultrashy ne particles as surrogatesfor ambient particles

2 Studies with ultramacrne particles from polymer thermodegradation

(a) Characterization and toxicity of inhaled polytetradeg uoroethylene (PTFE)

Heating of PTFE (Teregon) to ca 480 macrC results in the generation of fumes thatconsist of ultrashy ne particles and some gas-phase products mainly HF Heating totemperatures above 500 macrC will generate additional gas-phase products like perregu-oroisobutylene and others which are highly toxic (Lee amp Seidel 1991 Waritz ampKwon 1968 Makulova 1965) Studies in our laboratory were performed at heatingtemperatures below 500 macrC to avoid confounding of the responses due to the toxicgas-phase components The ultrashy ne particle size of the PTFE fumes ranges between

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Toxicology of ultramacrne particles in vivo studies 2721

20

05

15

00 00

10

20

30

40

500diameter (nm)

Ncm

ndash3 (

105 ) N

cmndash3 ( acute 10

4)

10 50

CMD = 1324 nm

agedfresh

GSD = 16

CMD = 203 nmGSD = 15

100

10

Figure 1 Particle size distribution of freshly generated and aged PTFE fumes showing the shiftfrom ultramacrne particle distribution to a size distribution with a median greater than 100 nm dueto coagulation after 35 min of ageing

10 and 50 nm Exposure of rats to a concentration of 5 pound 105 particles cmiexcl3 (equiva-lent to ca 50 m g miexcl3) for 1020 min results in the development of severe pulmonaryinregammation and hemorrhage within 4 h post exposure (Oberdorster et al 1995)High mortality due to the severe pulmonary oedematous response also occurs Lunglavage reguid shows signishy cantly increased neutrophils up to 80 of total cells highlyincreased protein levels and increases in lysosomal and cytoplasmic enzymes His-tologically a high degree of alveolar and interstitial oedema is present and bothepithelial and endothelial cell layers are severely disrupted These ultrashy ne particlesare thought to be the cause of the toxicity of PTFE fumes Using electron energy-loss spectroscopy (EELS) ultrashy ne reguorine-containing particles can be found shortlyafter the exposure in epithelial interstitial and endothelial sites of both the conduct-ing airways and alveolar regions of the lung (Oberdorster et al 2000) Analysis oflung mRNA shows signishy cant upregulation of proinregammatory cytokines such asIL-6 IL-1b IL-1a TNFnot and of antioxidants such as MnSOD and metallothioneinand the chemokine MIP-2 These responses are consistent with injuries due to severeoxidative lung damage

(b) Ereg ect of ageing on PTFE toxicity

Particles present in the air at high number concentrations tend to coagulate andthereby form larger particles over time This coagulation process can occur withinfractions of a second if number concentrations exceed 108 particles cmiexcl3 and willbe progressively slower at lower concentrations (Hinds 1982) Ageing of the freshlygenerated PTFE fumes for 35 min results in a shift of the particle size distributionfrom a count median diameter (CMD) of 1520 nm to one above 100 nm (shy gure 1)Gas-phase products do not seem to change in this ageing process If the toxicityof PTFE fumes is indeed caused by the presence of ultrashy ne particles and if larger

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2722 G Oberdorster

80

60

20

40

0

6

2

4

0agedfreshsham

P

MN

protein (mg

ml ndash1)

PMNsprotein

Figure 2 Lung lavage parameters of rats 4 h after a 50 min exposure to fresh and aged PTFEfumes The percentage of neutrophils of the total lavage cells and the lavage protein content areshown (N = 4 per group mean sectSD curren signimacrcantly direg erent from sham and aged (ANOVAp lt 005))

particles are less toxic one would predict that exposure to the aged PTFE fumeswith the larger particles will result in signishy cantly less toxicity Figure 2 shows theresults of a study in which rats were exposed to either the freshly generated PTFEfumes at a concentration of 5 pound 105 particles cmiexcl3 or to aged PTFE fumes at aconcentration of 15 pound 105 particles cmiexcl3 The mass concentrations of the particleswere 50 and 70 m g miexcl3 respectively Control rats were separately exposed to shy lteredair and inregammatory lung lavage parameters were determined 4 h post exposureAs can be seen from shy gure 2 the rats exposed to the freshly generated fumes showedthe expected high increase in lavage neutrophils and protein indicating the presenceof severe inregammatory pulmonary oedema In contrast sham-exposed control ratsand rats exposed to the aged fumes for 15 min did not show signishy cant changes inthe lavage parameters

Although these data are consistent with our hypothesis that inhaled ultrashy ne par-ticles have a signishy cantly greater toxic and inregammatory potential than larger sizedparticles it cannot be excluded that the ageing and coagulation of the fresh PTFEfumes for several minutes had altered potential short-lived toxic radicals on the sur-face of the particles (Pryor et al 1990) Whether the existence of such radicals playsa role in the toxicity of freshly generated polymer fumes is uncertain earlier stud-ies have shown that fumes generated from dinoterent plastic materials all show hightoxicity regardless of the presence or absence of short-lived radicals (Seidel et al 1991)

(c) Adaptation to PTFE fume toxicity

Workplace exposures to fumes of metals such as zinc or cadmium are known toinduce a state of tolerance upon repeated exposures (Drinker et al 1927) Such

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Toxicology of ultramacrne particles in vivo studies 2723

80

100

60

20

40

0

6

2

4

0non-adapted

(all died within 3 hours)n = 6

adaptedn = 6

shamn = 5

P

MN

protein (mg

ml ndash1)

8

PMNprotein

Figure 3 Lavage parameters 4 h after a 15 min exposure to PTFE fumes in adapted andnon-adapted F-344 rats The percentage neutrophils and total lavage protein content are shown(N = 6 per group mean sectSD curren signimacrcantly direg erent from sham and adapted group (ANOVAp lt 005))

adaptation is associated with increases of antioxidant proteins including metalloth-ionein (Hart et al 1989) which protect the lung from the toxic enotects of subsequentexposure to high concentrations of these fumes Such adaptive responses have notbeen demonstrated for polymer fumes However since the mechanism for the highacute toxicity of these metal fumes is most likely to be due to oxidative stress onemight expect that similar adaptive changes occur after exposures to polymer fumesconsisting of ultrashy ne particles

In order to investigate the induction of PTFE fume tolerance we exposed ratsto PTFE fumes containing 5 pound 105 particles cmiexcl3 (equivalent to ca 50 m g miexcl3) for5 min each on three consecutive days followed by a 15 min exposure to the sameconcentration on day four A second group of rats was sham exposed for 5 min onthree consecutive days and on day four received the 15 min PTFE fume exposuretogether with animals from group one A third group was sham exposed to shy lteredair on all four days Four hours after the 15 min exposure surviving animals wereeuthanized and their lungs lavaged for measurement of inregammatory parametersFigure 3 shows the result of this study with respect to lavage neutrophils and lavageprotein content During the three day adaptation animals of the adapted group

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2724 G Oberdorster

did not show any respiratory symptoms of being anotected by these short 5 minexposures Neither did the rats of this group show any clinical signs of respiratoryenotects after the 15 min exposure of PTFE fumes on day four In contrast rats of thenon-adapted group were severely anotected by the 15 min PTFE fume exposure on dayfour all of them had to be euthanized within 3 h post exposure because of severedyspnea Sham-exposed control animals like the adapted animals did not show anyenotects (shy gure 3)

The high inregammatory enotects in the lung observed after PTFE fume exposureare likely to also lead to systemic responses This in turn would anotect the ratsrsquobehaviour and work performance is expected to drop signishy cantly Indeed exposureof rats to PTFE fumes for 10 min was found to reduce performance on a runningwheel by up to 40 the enotect starting immediately after exposure and lasting severaldays However pre-exposure of rats on three consecutive days as was done in theadaptation study described above adapted the animals so that the 10 min PTFEexposure on day four did not lead to any reduction in work performance

The development of a state of tolerance against the deleterious enotects of ultra-shy ne PTFE fumes in these experiments demonstrates the importance of pre-exposurehistory for the induction of pulmonary and probably systemic responses This mayalso be important for ambient particles Adaptation is known to occur after expo-sure to oxidant gases such as ozone (Van Bree et al 1993 Dodge et al 1994) Suchprotective responses may be age dependent and adaptation may not be as completeor may take longer if the host organism is compromised and in a more susceptiblestate

(d ) Particle versus gas phase toxicity of PTFE fumes

Since heating of PTFE results in the generation of both ultrashy ne particles andgas-phase products the question arises as to whether the ultrashy ne particles per secan induce the observed highly toxic inregammatory responses In order to evaluatethis an experiment was performed with exposure of four groups of rats as follows

group 1 sham-exposed control animals

group 2 animals exposed to the complete phase (particle plus gas) of freshly gen-erated PTFE fumes

group 3 animals exposed to the gas phase only after shy ltering the ultrashy ne particlesand

group 4 animals exposed to ultrashy ne PTFE particles only

For the latter group the generation of PTFE fumes was changed from heating Teregonin air to heating in argon which resulted in the generation of ultrashy ne particles withno detectable gas-phase compounds The exposure was for 25 min in compartmental-ized whole-body exposure chambers at an ultrashy ne particle number concentration of5 pound 105 particles cmiexcl3 Four hours after exposure the animals were euthanized andinregammatory lung lavage parameters determined As shown in shy gure 4 neither thegroup exposed to the gas phase alone nor the group exposed to the ultrashy ne particlesalone showed signishy cant responses with respect to lavage neutrophils or protein com-pared with control animals In contrast the group exposed to the complete fume

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Toxicology of ultramacrne particles in vivo studies 2725

80

60

20

40

0

8

6

2

4

0particle (Ar)particle + gas gas phasesham

P

MN

protein (mg

ml ndash1)

PMNprotein

Figure 4 Lung lavage parameters of rats 4 h after a 25 min exposure to the gas or particle phaseof PTFE fumes or to the complete particle-plus-gas phase The percentage neutrophils of totallavage cells and lavage protein content are shown (N = 5 per group mean sectSE curren signimacrcantlydireg erent from all other groups (ANOVA p lt 005))

particle plus gas phase conshy rmed the highly toxic nature of the PTFE fumes byresponding with high increases in both lavage neutrophils and protein

This result indicates that `cleanrsquo ultrashy ne PTFE particles (generated in argon) donot induce signishy cant toxicity after short-term exposure However it is conceivablethat the argon-generated PTFE particles may have dinoterent surface reactivity com-pared with the air-generated PTFE particles which may contribute to their hightoxicity Clearly the gas-phase constituents alone when generated at below 500 macrCdo not exert pulmonary toxicity but their combination with the ultrashy ne particlesdoes Preliminary attempts to adsorb PTFE gas-phase constituents onto ultrashy necarbon particles did not result in acute pulmonary inregammatory responses Earlierstudies have also found that gas-phase compounds of PTFE fumes do not cause thehigh toxicity when generated below 500 macrC (Waritz amp Kwon 1968) However addi-tional studies may be needed to conshy rm that gas-phase compounds do not play arole in PTFE fume toxicity

(e) Conclusions from PTFE fume studies

The conclusions from these studies with PTFE fumes are that the ultrashy ne par-ticles are indeed key to their high pulmonary toxicity Furthermore coagulation tolarger particles during the ageing process results in a signishy cant loss of the high ultra-shy ne particle toxicity The translocation of ultrashy ne particles deposited in the lungto epithelial interstitial and endothelial sites also appears to be rapid conshy rmingearlier observations (Stearns et al 1994) This is probably due to the fact that these

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2726 G Oberdorster

8

7

5

6

010010101001

nuclei mode accumulation mode coarse mode

geometric diameter ( m m)

DV

Dlo

g D

p (m

3cm

ndash3)

0002

4

3

2

1

vapour

nucleation

coagulation

DGV = 0018

g = 16sDGV = 021

g = 18s

DGV = 49

g = 187s

condensation

mechanicallygenerated

fine-mode particles coarse-mode particles

Figure 5 Trimodal urban aerosol typical size distribution (from EPA 1996)

very tiny particles escape alveolar macrophage surveillance which is very emacr cientfor larger particles (Hahn et al 1977) Of signishy cance is that the very high acutetoxicity of ultrashy ne PTFE fumes can be prevented by short-term pre-exposuresleading to protective adaptation phenomena in the lung Whether these shy ndingscan be extrapolated to ultrashy ne particles of low toxicity such as those occurring inthe urban atmosphere requires studies with relevant ultrashy ne particles of low toxi-city The following sections show that some concepts of ultrashy ne particle toxicity doindeed apply to other ultrashy ne particles such as the relative potencies of ultrashy neand larger particles translocation across the epithelium and to interstitial sites andthe importance of combined exposure of ultrashy ne particles with an oxidant gas

3 Studies with ultramacrne particles of low toxicity

(a) Dosimetric aspects of ultramacrne particle toxicology

As mentioned in x 1 ultrashy ne particles are one of three particle modes in the urbanatmosphere (shy gure 5) Normal background levels of the ultrashy ne mode are below2 m g miexcl3 with number concentrations in the range 14 pound104 particles cmiexcl3 (Hugheset al 1998) Table 1 summarizes results of measurements which show that episodicevents can give rise to much higher concentrations approaching 1pound106 particles cmiexcl3

and mass concentrations of up to 50 m g miexcl3 In addition to tramacr c-related increasesother sources seem to be responsible for these high concentrations as well and fur-ther research is needed to determine the composition of these particles Episodicincreases in ultrashy ne particle concentrations occur frequently and can last for morethan a day and it is probably during or after these events that health enotects may

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Toxicology of ultramacrne particles in vivo studies 2727

Table 1 Urban atmosphere particle concentrations ultramacrnes (below 100 nm)

(Nature of particles carbonaceous and others)

condition particles (cm iexcl 3 ) ( m g m iexcl 3 ) reference

background 15 pound104 0816 Tuch et al (1997)

average (Erfurt Hughes et al (1998)

Los Angeles) (Cass 10 t diexcl 1 emitted

in Southern California)

episodic event 3 pound 105 ca 50 Brand et al (1992)

peak (Frankfurt)

episodic events and 1 pound 106 P McMurry

traplusmn c peak (Atlanta) (2000 personal communication)

(preliminary data)

occur Therefore these concentrations can serve as a guideline for designing toxico-logical studies using realistic exposure levels Importantly when performing studieswith ultrashy ne particle exposures in experimental animals one should attempt toestimate deposited lung doses of ultrashy ne particles with the goal of achieving lungburdens that would also be experienced by humans under a given urban exposurescenario Exposure concentrations to achieve this may be dinoterent between humansand experimental animals as is discussed below

Deposition of inhaled ultrashy ne particles in the respiratory tract occurs by dinotu-sional processes Several studies have shown that the human nose is highly emacr cientin collecting inhaled ultrashy ne particles by this process in the nasal compartment(Cheng et al 1991 Swift et al 1992) However these data also show that dinotusionaldeposition in the nasopharyngeal compartment is highly dependent on the ultrashy neparticle size such that ultrashy ne particles below ca 10 nm deposit with high emacr -ciency in the nose whereas this deposition becomes less for particles between 10 and100 nm There is thus a misconception that all ultrashy ne particles deposit emacr cientlyin the nose However predictive particle deposition models show that the probabilityof deposition of 20 nm particles is greatest in the alveolar region (shy gure 6) Likewisethe tracheobronchial region can also be a signishy cant target for even smaller ultrashy neparticles as indicated in shy gure 6 If the deposited dose is expressed per unit surfacearea of the epithelium the tracheo-bronchially deposited dose of ultrashy ne particlescan be up to 50-fold higher than that for the alveolar region These are importantdosimetric aspects in ultrashy ne particle toxicology which need to be considered whenstudying these particles In addition an important factor is the fate of depositedultrashy ne particles that is their disposition which seems to be dinoterent from largerparticles as was discussed in the PTFE fume experiments

Another important aspect of particle toxicology in general relates to comparativedosimetry between experimental animals and humans Rodents as the most fre-quently used experimental animals have dinoterent deposition emacr ciencies for particlesin their respiratory tract compared with humans Table 2 compares the depositionemacr ciency of inhaled 20 nm particles|which is the peak of the urban ultrashy ne particlemode|between rats and humans based on deposition models by Yeh amp Schum (1980)for rats and the ICRP (1994) model for humans As this table indicates the humanequivalent concentration for 20 nm ultrashy ne particles is about twice that for the rat

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2728 G Oberdorster

10

08

04

02

06

00

100101010010001

diameter (m m)

r

egio

nal d

epos

itio

n

00001

total

NPL

A

TB

Figure 6 Predicted deposition of inhaled particles of direg erent sizes of unit density in the humanrespiratory tract during nose breathing light exercise (ICRP 1994) NPLrsquo denotes nasopharyn-golaryngeal deposition TBrsquo denotes tracheobronchial deposition Arsquo denotes alveolar deposi-tion totalrsquo denotes the sum of particle depositions in the respiratory tract for all three com-partments

Table 2 Particle dosimetry

(Human equivalent concentration for inhaled 20 nm particles (ICRP human model and Yeh ampSchum rat model))

alveolar deposited dose over relative dosedeposition 6 h per m2 alveolar at same inhaled

fraction surface at 10 m g m iexcl 3 concentration

rat nasal breathing at rest 027 365 ng 1

human nasal breathing light 05 692 ng 19

The aforementioned high episodic ultrashy ne particle concentration of 50 m g miexcl3 inurban air would therefore be equivalent to ca 100 m g miexcl3 inhaled by rats Sincethis comparison is based on predictive deposition models and since these modelshave not been validated experimentally for ultrashy ne particles there could be con-siderable uncertainties For example signishy cant dinoterences exist between the NCRPand the ICRP deposition model Thus there is an urgent need to validate thesedeposition models by experimental data in rodents as well as humans

(b) Indeg ammatory potential of ultramacrne versus macrne particles

We postulate that deposited ultrashy ne particles induce a greater inregammatoryresponse per given mass than larger particles of the size of the accumulation mode

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Toxicology of ultramacrne particles in vivo studies 2729

40

20

10

30

0 200015001000500

particle mass ( m g)

PMN

sultrafine TiO2fine TiO2saline

Figure 7 Indeg ammatory response 24 h after instillation of direg erent doses of ultramacrne and macrneTiO2 in rats expressed as percentage neutrophils in lung lavage as a function of instilled particlemass Ultramacrne TiO2 ca 20 nm macrne TiO2 ca 250 nm

40

20

10

30

0 25015010050

particle surface area (cm2)

P

MN

s

200

ultrafine TiO2fine TiO2saline

Figure 8 Same data as shown in macrgure 7 with particle dose expressed asparticle surface area in the lung

Results of our studies with ultrashy ne freshly generated and larger aged PTFE fumesdescribed above are consistent with this hypothesis We tested this hypothesis bydosing rats with two dinoterent particle types of a rather benign dust TiO2 Ultrashy ne

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Table 3 Accumulation versus nucleation (ultramacrne) mode particlespulmonary indeg ammatory potential in humans

(Assumptions composition of two particle types is the same toxicity is proportional to depositeddose expressed as particle surface area (example macrne and ultramacrne TiO2 ))

accumulation mode particle ultramacrne particle(ca 250 nm) (ca 20 nm)

relative alveolar deposition 1 36

relative particle surface area 1 10

relative predicted toxicity 1 36a

(10 m g m iexcl 3 ultramacrne sup2 360 m g m iexcl 3 accumulation mode)

a Additional factors need to be considered increased interstitial translocation leads to extrapul-monary ereg ects

TiO2 with an average particle size of 20 nm and pigment grade (shy ne) TiO2 with anaverage particle size of ca 250 nm were used Doses ranging from 30 to 2000 m g ofTiO2 were intratracheally instilled into groups of rats The inregammatory response intheir lungs was assessed by analysis of cellular and biochemical lung lavage param-eters 24 h later The result of this doseresponse study is shown in shy gure 7 It isevident from this shy gure that ultrashy ne TiO2 elicited a signishy cantly greater inregam-matory cell inregux (neutrophils) for the same dose than larger sized TiO2 Howeverwhen the deposited TiO2 dose was expressed as particle surface area the result wasquite dinoterent as shown in shy gure 8 Using the particle surface area as dosimetricresulted in virtually identical inregammatory responses of these two dinoterent sizes ofTiO2 particles The importance of particle surface area for eliciting inregammatoryresponses in the lung has been conshy rmed by Li et al (1996) with ultrashy ne and shy necarbon-black particles This concept of particle surface area as the appropriate dosi-metric has been recognized as an important principle in particulate matter toxicology(Oberdorster 1996 Donaldson et al 1998)

Considering dinoterences in pulmonary deposition and the importance of dosimetricfor characterizing the inregammatory potential of inhaled particles one can deduce arelative potency ranking for the in vivo toxicity of inhaled ultrashy ne particles versuslarger 250 nm particles of the accumulation mode Assuming that the chemical com-position of the two particle sizes is the same and that the toxicity is proportional tothe deposited dose expressed as particle surface area one can derive that the tox-icity of ultrashy ne particles is about 36-fold greater than that of accumulation-modeparticles in terms of the inhaled mass concentration Table 3 shows that this fac-tor is due to the 36-fold greater deposition emacr ciency in the alveolar region and theten-fold larger particle surface area per given mass for the 20 nm particles comparedwith 250 nm particles Additional factors may need to be considered such as thedinoterence in interstitial translocation between the two particle sizes and possiblyalso dinoterences in translocation to extrapulmonary sites

As mentioned above TiO2 particles are of a rather benign nature and have beenused in the past in a number of studies as control particles of low toxic potencyagainst which enotects of other particle types have been compared It is likely that theinregammatory response elicited by higher doses of ultrashy ne TiO2 is based on a similaroxidative stress mechanism to that underlying the much greater pulmonary toxicity

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Toxicology of ultramacrne particles in vivo studies 2731

25

15

5

10

0+PTFE

ndashPTFEshamexposure

P

MN

in B

AL

20

Figure 9 Lavage neutrophil response in rats 24 h after intratracheal instillation of 100 m g ofTiO2 particles in PTFE-fume-adapted and non-adapted rats curren signimacrcantly direg erent groupswithout PTFE and sham exposure (ANOVA p lt 005)

of PTFE fumes discussed above (Donaldson et al 1998) One might therefore expectthat adaptive responses observed in our PTFE experiments would also attenuate theinregammatory response of ultrashy ne TiO2 based on the existence of cross-toleranceThe result of a study in rats indeed showed a signishy cantly reduced inregammatoryresponse in the lung to intratracheally instilled 100 m g of TiO2 when the animals hadbeen adapted to PTFE fumes for the previous three days (shy gure 9)

(c) Deposition studies with ultramacrne carbon and other particles

A major component of ambient particles generated by combustion processes istheir carbonaceous core (Hughes et al 1998) These particles consist of dinoterentinorganic and organic compounds and we used carbonaceous particles consisting ofelemental and ca 30 organic carbon in our initial studies as surrogates to determinewhether these ultrashy ne particles can induce enotects in the lung We used an electricspark discharge system that generates ultrashy ne carbonaceous particles between twographite electrodes in an argon atmosphere Figure 10 shows a typical particle sizedistribution with a count median diameter of 24 nm and a geometric standard devi-ation (GSD) of 186 One of our goals was to determine the fate of these ultrashy neparticles in the lung In collaboration with Dr Godleski (Harvard University) usingEELS technology we could show that these ultrashy ne carbonaceous particles werepresent in type I and type II alveolar epithelial cells shortly after a 6 h exposure toca 100 m g miexcl3

In order to further evaluate whether ultrashy ne particles after deposition can alsotranslocate to extrapulmonary tissues we used ultrashy ne platinum particles (CMD13 nm GSD 17) and exposed a rat to these particles for 6 h at a concentration of

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2732 G Oberdorster

40

30

10

20

00

100diameter (nm)

count median 241 nm GSD 186

Ncm

ndash3 (acute

105 )

10

Figure 10 Particle size distribution of ultramacrne carbonaceous particles generated by electricspark discharge between graphite electrodes in an argon atmosphere

70

60

10

50

40

30

20

0

50001000100endotoxin units (EU) deposited

P

MN

in B

AL

10

Figure 11 Doseresponse relationship of lung lavage neutrophils 24 h afterdireg erent lung doses of inhaled endotoxin in young rats

ca 100 m g miexcl3 With the use of inductively coupled plasma mass spectroscopy plat-inum levels were determined 30 min after the exposure in dinoterent lobes of the lungthe trachea and the liver A total of 212 m g of platinum was found to be deposited in

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Toxicology of ultramacrne particles in vivo studies 2733

20

15

5

10

0

TiO2

P

MN

LPS+ TiO2

ultrafine

LPS TiO2

LPS+ TiO2

fine

LPScontrol

Figure 12 Lung lavage neutrophils 24 h after intratracheal instillation of 50 m g of ultramacrne(20 nm) or macrne (250 nm) TiO2 particles into rats with or without prior LPS priming comparedwith LPS alone and saline-instilled control rats (mean sectSE) curren signimacrcant direg erence to con-trol rats currencurren signimacrcant direg erence to LPS-primed group and to ultramacrne particle only group(P lt 005 one-way ANOVA)

the lower respiratory tract which corresponds to an estimated deposition emacr ciencyof 20 of the inhaled ultrashy ne platinum particles A signishy cant shy nding was thatplatinum was also found in the liver which amounted to ca 7 of the lung platinumburden However it would be premature to conclude that this indicates transloca-tion of the ultrashy ne particles from the lung since it cannot be excluded that a smallamount of the ultrashy ne platinum particles has been solubilized in the lung and mayhave reached the liver as soluble platinum Thus although platinum metal is consid-ered to be very poorly soluble additional studies with insoluble ultrashy ne particlesneed to be performed and are planned in our laboratory to determine more preciselythe potential for ultrashy ne particles to reach extrapulmonary tissues

(d ) Animal models of a compromised host to study ultramacrne particle toxicity

Associations between particulate air pollution and adverse health enotects have onlybeen observed in susceptible parts of the population and not in healthy people Animportant aspect of studying potential causality of such enotects includes thereforethe use of animal models which mimic a compromised respiratory or cardiovascularcondition occurring in humans Inhalation studies with particles in the past weretypically performed in young healthy animals using high exposure concentrationsand doses in order to induce enotects that can then be analysed further With increasingawareness of dosimetry issues (low relevant doses) and of the importance of theimpact of diseases on enotects those previous study designs have to be questionedare mechanisms underlying enotects induced by high doses in the healthy mammalian

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2734 G Oberdorster

carbonozone

ozone

carbon

control(a)

young

LPS

LPSozone

LPScarbon

LPScarbonozone

0 10

PMN

lavage neutrophils

20 30

old

Figure 13 Lung lavage indeg ammatory cell response of young (10 weeks) and old (22 month) ratsfollowing inhalation exposure to ultramacrne carbonaceous particles (ca 105 m g m iexcl 3 ) sect O3 (1 ppm)sect LPS priming (a) Per cent neutrophils of total lavage cells

organisms really the same as those of low doses in a compromised host Or shouldwe not rather assume that the dosedose rate controls the mechanism as has beenconcluded from a number of particle overload studies

A change in particulate matter toxicology is taking place switching from the useof healthy animals to that in animal models of compromised humans These modelswhich need to be characterized and validated include specishy c disease models the useof transgenic animals and of senescent animals In addition as already emphasizedrelevant realistic doses both under in vivo and in vitro experimental study conditionsneed to be applied For example in the aforementioned studies with intratracheallyinstilled ultrashy ne and shy ne TiO2 particles high doses were administered that will notbe deposited by inhalation of low ambient concentrations in short-term exposuresHowever the goal of those TiO2 instillation studies was to test the concept of therelative toxicities of ultrashy ne versus shy ne particles rather than examining whetherultrashy ne particles at reported ambient concentrations can cause adverse enotects

The critical issue of appropriate animal models of a human disease is complicatedby the fact that many animal models are of an acute nature whereas respectivehuman conditions have slowly developed into a chronic state For example intra-tracheal instillation of elastase produces a marked pulmonary emphysema in miceor rats yet this emphysema is most certainly not equivalent to human emphysema

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Toxicology of ultramacrne particles in vivo studies 2735

carbonozone

ozone

carbon

control(b)

LPS

LPSozone

LPScarbon

LPScarbonozone

0 40

AUC

PMA-stimulatedchemiluminescence

80 120

Figure 13 (Cont) (b) PMA-stimulated chemiluminescence of lavage cells

seen in people with chronic obstructive pulmonary disease In our initial studieswith a compromised respiratory tract we used an inhalation model with endotoxin(lipopolysaccharide (LPS)) to mimic the early stages of a respiratory tract infectionwith gram negative bacteria People with pneumonia in particular the elderly areone susceptible group that has been identishy ed in epidemiological studies (EPA 1996)which we are targeting in our studies

Figure 11 shows the doseresponse relationship of inhaled LPS in rats 24 h afterexposure The neutrophil response in lung lavage reguid after dinoterent doses of endo-toxin deposited in the alveolar region is shown The LPS doses were estimated basedon the particle size distribution of the inhaled LPS aerosol and the airborne con-centration with the use of predictive deposition models (Yeh amp Schum 1980) LPSexposure lasted only for ca 12 min Depending on the deposited dose LPS at veryhigh doses can result in a severe ARDS-like pulmonary inregammation with largeamounts of neutrophils and protein in the lavage reguid However at lower doses onlya mild inregammatory response in terms of neutrophil inregux and no increase in lavageprotein occurs We used this lower dose of ca 70 endotoxin units (EUs) to prime therespiratory tract prior to exposure with ultrashy ne particles The mild inregammatoryresponse at this low deposited alveoles dose is characterized by a lavage neutrophillevel of ca 10 of the total cells 24 h post exposure

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2736 G Oberdorster

Before using this LPS model to examine the response to inhaled low concentra-tions of spark discharge-generated ultrashy ne carbonaceous particles we tested it usingultrashy ne and shy ne TiO2 particles via intratracheal instillation 50 m g of ultrashy ne andshy ne TiO2 were intratracheally instilled in rats that had either received the LPS prim-ing inhalation or a sham inhalation of NaCl aerosol Instillation of the particles wasperformed within 30 min after the inhalation Other rats received inhaled LPS aloneor instilled saline (controls) As the result in shy gure 12 shows only the ultrashy ne TiO2

particles given after LPS induced a signishy cantly greater neutrophil inregux comparedwith TiO2 alone and LPS alone whereas the shy ne TiO2 particles administered tothe LPS-primed lung did not show a greater response than LPS alone This resultshows that priming of the respiratory tract with inhaled LPS can indeed amplifythe response to a subsequent particulate stimulus and it further conshy rms that forthe same lung dose in terms of mass ultrashy ne particles are signishy cantly more potentthan shy ne particles

(e) Age and ozone co-exposure as modulators of ultramacrne carbon particle toxicity

The senescent mammalian organisms may be more sensitive to inhaled toxicantsthan the younger organism With respect to the potential enotect of ambient partic-ulate matter it has been suggested that co-exposure to other pollutants such asoxidant gases may contribute to the adverse enotects observed in the epidemiologicalstudies (Burnett et al 1997a b Samet et al 1997) Our studies with ultrashy ne PTFEfumes although inconclusive with respect to a contributory enotect of gas-phase com-pounds can be interpreted as showing some inreguence of gas-phase compounds Ourlatest study with inhaled ultrashy ne carbonaceous particles was designed to evaluatetheir inregammatory enotects in the lung of young and old rats with and without ozoneco-exposure and with and without LPS inhalation priming Eight groups of 10-weekold and 22-month old rats were exposed to ultrashy ne carbonaceous particles (concen-tration ca 105 m g miexcl3) ozone (1 ppm) or inhaled LPS (ca 70 EU estimated alveolardose) and to combinations of these compounds Sham-exposed control rats served ascontrols Lung lavage parameters were determined 24 h later and lavaged inregamma-tory cells were subjected to a chemiluminescence assay in vitro to determine theirunstimulated and phorbol ester (PMA)-stimulated oxidant release

Figure 13 shows the result with respect to lavage neutrophils and the PMA-stimulated chemiluminescence Three-way analyses of variance (ANOVAs) for thegroups of young and old rats as well as a four-way ANOVA for the two age groupscombined were performed the three factors were ultrashy ne carbon ozone and LPSand the fourth factor was age These analyses showed that each of the three compo-nents (ultrashy ne carbon ozone and LPS) induced signishy cant enotects independently Inaddition the ultrashy ne carbonaceous particle response in the aged rats was synergisticwith the enotects of ozone In both old and young groups the greatest inregammatorycell response was observed in the LPS-primed group with combined exposure toultrashy ne carbonaceous particles and ozone (Elder et al 2000b)

There was also a signishy cant age enotect showing that the aged animals respondedwith greater oxidant release of the lavaged inregammatory cells compared with theyoung animals in the combined exposure groups This greater release of reactiveoxygen species implies a greater risk of oxidative lung injury in the aged organismunder exposure conditions of ultrashy ne carbon particles in combination with ozone in

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Toxicology of ultramacrne particles in vivo studies 2737

the LPS-sensitized respiratory tract These studies show that ultrashy ne carbonaceousparticles can cause signishy cant pulmonary inregammation This occurs at inhaled con-centrations leading to lung doses which are deposited in human lungs during episodicincreases of urban ultrashy ne particles Future studies will evaluate whether additionof transition metals to the carbon particles amplishy es the inregammatory response

4 Summary and conclusions

Conclusions from these studies are as follows

(1) Poorly soluble ultrashy ne particles cause a signishy cantly greater pulmonary in-regammation per given mass than larger particles The appropriate dosimetricis their high specishy c surface area rather than the mass of these particles

(2) Ultrashy ne carbonaceous particles at relevant inhaled concentrations can causean inregammatory response in rodents

(3) Ultrashy ne particles translocate readily to epithelial and interstitial sites It isalso conceivable that they may be transported to extrapulmonary organs thisneeds to be conshy rmed in future studies

(4) Specishy c modulating factors that increase ultrashy ne particle enotects include ageand a compromisedsensitized respiratory tract

(5) Combined exposures with an oxidant gas can enhance ultrashy ne particle enotects

Studies with ultramacrne particles mentioned in this manuscript have been supported by the HealthEreg ects Institute (contract no 95-11) and by the National Institute of Health Sciences grants(ES 04872 and ES 247) Detailed results of these studies were reported by Elder et al (2000ac)Johnston et al (1996 1998) and Oberdorster et al (1995 2000)

References

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Burnett R T Brook J R Yung W T Dales R E amp Krewski D 1997a Association betweenozone and hospitalization for respiratory diseases in 16 Canadian cities Environ Res 72 2431

Burnett R T Cakmak S Brook J R amp Krewski D 1997b The role of particulate size andchemistry in the association between summertime ambient air pollution and hospitalizationfor cardiorespiratory diseases Environ Health Perspect 105 614620

Cheng Y-S Yeh H-C amp Swift D L 1991 Aerosol deposition in human nasal airway forparticles 1 nm to 20 nm a model study Radiation Protection Dosimetry 38 4147

Dodge D E Rucker R B Pinkerton K E Haselton C J amp Plopper C G 1994 Dose-dependent tolerance to ozone III Elevation of intracellular clara cell 10-k protein in centralacini of rats exposed for 20 months Toxicol Appl Pharmacol 127 109123

Donaldson K Li X Y amp MacNee W 1998 Ultramacrne (nanometer) particle mediated lunginjury J Aerosol Sci 29 553560

Drinker P Thomson R M amp Finn J L 1927 Metal fume fever II Resistance acquired byinhalation of zinc oxide on two successive days J Ind Hygiene Toxicol 9 98105

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2738 G Oberdorster

Elder A C P Johnston C Finkelstein J amp Oberdorster G 2000a Induction of adaptationto inhaled lipopolysaccharide in young and old rats and mice Inhal Toxicol 12 225243

Elder A C P Gelein R Finkelstein J Cox C amp Oberdorster G 2000b Endotoxin primingareg ects the lung response to ultramacrne particles and ozone in young and old rats Inhal Toxicol(Suppl I) 12 8598

Elder A C P Gelein R Finkelstein J Cox C Oberdorster G 2000c The pulmonaryindeg ammatory response to inhaled ultramacrne particles is modimacred by age respiratory tractsensitization and disease Inhal Toxicol (In the press)

EPA 1996 Air quality criteria for particulate matter vol III EPA600P-95001cF

Goldstein M Weiss H Wade K et al 1987 An outbreak of fume fever in an electronicsinstrument testing laboratory J Occup Med 29 746749

Gordon T Chen L C Fine J M Schlesinger R B Su W Y Kimmel T A amp AmdurM O 1992 Pulmonary ereg ects of inhaled zincoxide in human subjects guinea pigs rats andrabbits Am Ind Hygiene Ass J 53 503509

Hahn F F Newton G J amp Bryant P L 1977 In vitro phagocytosis of respirable-sizedmonodisperse particles by alveolar macrophages In Pulmonary macrophages and epitheliacells (ed C L Sanders R P Schneider G E Dagle amp H A Ragen) pp 424435 EnergyResearch and Development Administration Symposium Series

Hart B A Voss G W amp Willean C L 1989 Pulmonary tolerance to cadmium followingcadmium aerosol pretreatement Toxicol Appl Pharmacol 101 447460

Hinds W C 1982 Aerosol technology pp 235239 Wiley

Hughes L S Cass G R Jones J Ames M amp Olmec L 1998 Physical and chemicalcharacterization of atmospheric ultramacrne particles in the Los Angeles area Environ SciTechnol 32 11531161

ICRP (International Commission on Radiological Protection) 1994 Annals of the ICRP humanrespiratory tract model for radiological protection ICRP publication 66 Oxford Pergamon

Johnston C J Finkelstein J N Gelein R Baggs R amp Oberdorster G 1996 Characterizationof the early pulmonary indeg ammatory response associated with PTFE fume exposure ToxicolAppl Pharmacol 140 154163

Johnston C J Finkelstein J N Gelein R M amp Oberdorster G 1998 Pulmonary indeg am-matory responses and cytokine and antioxidant mRNA levels in the lungs of young and oldC57BL6 mice after exposure to Tedeg on fumes Inhal Toxicol 10 931953

Lee K P amp Seidel W C 1991 Pulmonary response to perdeg uoropolymer fume and particlesgenerated under various exposure conditions Fund Appl Toxicol 17 254269

Li X Y Gilmour P S Donaldson K amp MacNee W 1996 Free radical activity and pro-indeg ammatory ereg ects of particulate air pollution (PM1 0 ) in vivo and in vitro Thorax 5112161222

Makulova I D 1965 The clinical picture in acute perdeg uoroisobutylen poisoning Gigiena TrudaI Professionalnye Zabolevaniya 9 2023

Oberdorster G 1996 Signimacrcance of particle parameters in the evaluation of exposuredoseresponse relationships of inhaled particles Inhal Toxicol (Suppl) 8 7389

Oberdorster G Gelein R M Ferin J amp Weiss B 1995 Association of particulate air pollutionand acute morality involvment of ultramacrne particles Inhal Toxicol 7 111124

Oberdorster G Finkelstein J N Johnston C Gelein R Cox C Baggs R amp Elder A2000 Investigatorrsquo s report acute pulmonary ereg ects of ultramacrne particles in rats and miceHealth Ereg ects Institute macrnal report (In the press)

Pryor W A Nuggehalli S K Scherer Jr K V amp Church D F 1990 An electron spinresonance study of the particles produced in the pyrolysis of perdeg uoro-polymers Chem ResToxicol 3 27

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Discussion

M S Bingley (Cobham Surrey UK ) The European Community is about toremove lead (Pb) from solder Replacement solders will have a higher melting pointElectronic circuitry makes extensive use of PTFE In view of what you have saidabout the toxicity of ultrashy ne particles generated by heated PTFE are electronicengineers to be put in danger by future EC legislation

G Oberdorster The particles in fumes from solder seem to be bigger than ultra-shy nes and you can actually see those fumes in contrast to PTFE fumes If PTFEis present on electronic circuitry and is heated at the same time by the solder theemitted ultrashy ne PTFE particles will most likely coagulate onto the larger particlesof the dense solder fume Experiments adding PTFE fumes to diesel smoke or woodsmoke have shown that these combined particles were 80 times less potent in theirtoxicity than PTFE fumes

D Costa (US EPA NC USA) Your last shy gure showing the combined enotectsof ultrashy nes ozone and LPS showed that 100 m g miexcl3 of ultrashy nes alone had littleimpact on PMNs and likewise the ozone exposure also had little enotect where manypublished results show a signishy cant enotect Yet the interaction (combined) enotectseems larger Do you have an explanation for this

G Oberdorster Indeed the response of neutrophils in BAL was rather low afterozone alone and after ultrashy ne carbon alone However the combined exposure in the

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2740 G Oberdorster

LPS-primed animals showed a large response which emphasizes the importance ofestablishing animal models of increased susceptibility for evaluating otherwise subtleenotects of inhaled ultrashy ne carbon I think that the development and use of specishy canimal models|with a compromised pulmonary or cardiovascular system|is crucialfor future progress in PM research

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