Costa Bioavailability Metals

Bioavailable Transition Metals in Particulate (3,5,6). Subsequent studies have suggested
that certain individuals may be at higher
Matter Mediate Cardiopulmonary Injury in risk for adverse effects of PM (3,7-10).
Healthy and Compromised Animal Models The elderly with chronic cardiopulmonary
diseases, those with pneumonias, and
those with asthma at any age appear to be
Daniel L. Costa and Kevin L. Dreher at higher risk. However, considerable
uncertainty remains about specific bio-
Pulmonary Toxicology Branch, Experimental Toxicology Division,
markers or biological mechanism(s) that
National Health and Environmental Effects Research Laboratory,
might underlie the higher risk.
Research Triangle Park, North Carolina Although the epidemiology of ambient
air PM is compelling, these findings suffer
Many epidemiologic reports associate ambient levels of particulate matter (PM) with human
from the limited ability of epidemiology
mortality and morbidity, particularly in people with preexisting cardiopulmonary disease (e.g.,
to address the issue of causality. However,
chronic obstructive pulmonary disease, infection, asthma). Because much ambient PM is derived
animal toxicology studies, though encum-
from combustion sources, we tested the hypothesis that the health effects of PM arise from
bered with other limitations that relate to
anthropogenic PM that contains bioavailable transition metals. The PM samples studied derived
extrapolation, are capable of directly
from three emission sources (two oil and one coal fly ash) and four ambient airsheds (St. Louis,
addressing questions regarding the mecha-
MO; Washington; Dusseldorf, Germany; and Ottawa, Canada). PM was administered to rats by
nisms of response to PM with controlled
intratracheal instillation in equimass or equimetal doses to address directly the influence of PM
empirical study designs. We used a toxico-
mass versus metal content on acute lung injury and inflammation. Our results indicated that the
logic approach to address two critical
lung dose of bioavailable transition metal, not instilled PM mass, was the primary determinant of
questions that have evolved from the
the acute inflammatory response for both the combustion source and ambient PM samples.
epidemiology database:
Residual oil fly ash, a combustion PM rich in bioavailable metal, was evaluated in a rat model of
cardiopulmonary disease (pulmonary vasculitis/hypertension) to ascertain whether the disease * Are there constituents common to PM
state augmented sensitivity to that PM. Significant mortality and enhanced airway that could account for the consistency
responsiveness were observed. Analysis of the lavaged lung fluids suggested that the milieu of in epidemiologic findings across such
the inflamed lung amplified metal-mediated oxidant chemistry to jeopardize the compromised diverse exposure environments?
cardiopulmonary system. We propose that soluble metals from PM mediate the array of * Can animal models of cardiopulmonary
PM-associated injuries to the cardiopulmonary system of the healthy and at-risk compromised disease elucidate the etiology of appar-
host. Environ Health Perspect 1 05(Suppl 5):1053-1060 (1997) ent subpopulation susceptibilities to
PM as identified by epidemiology?
Key words: particulate matter, metals, air pollution, lung injury, animal models, pulmonary
These two questions transcend the PM
hypertension
issue and focus attention on the underlying
causation and toxicologic potency of ambient
air PM and how the physicochemical proper-
Introduction ties of PM link to the apparent potentiation
Over the last several years, a robust estimates across diverse urban airsheds, ofeffects in predisposed individuals.
database has emerged from a series of epi- based primarily on outdoor mass metrics of Ambient air PM1o is comprised of a
demiology studies demonstrating a statisti- exposure, i.e., total suspended particulates complex mixture of crustal and anthro-
cal association between ambient air (TSP) and PM.O pm in aerodynamic pogenic matter that are naturally separated
diameter (PM1o) (3,4). Indeed, as the mass
pollution particulate matter (PM) and by size. The crustal material, consisting
mortality/morbidity among the exposed metric for PM is restricted to smaller size largely of insoluble silicates derived from
human population (1-4). These studies ranges, the strength of the statistical corre- abrasive processes, is typically larger in size
lation increases (i.e., PM2 5 > PM1o > TSP)
revealed remarkably consistent unit risk (. 2.5 pm) and often is referred to as coarse
mode PM. The PM fraction below 2.5 pm
is considered fine mode; it arises from com-
This paper is based on a presentation at The Sixth International Meeting on the Toxicology of Natural and Man- bustion processes or atmospheric transfor-
Made Fibrous and Non-Fibrous Particles held 15-18 September 1996 in Lake Placid, New York. Manuscript
mation of combustion emissions. The PM
received at EHP26 March 1997; accepted 17 July 1997.
fine mode is of particular concern as a
The authors acknowledge the contributions of the staff of the Pulmonary Toxicology Branch in this research
potential health threat. As this fraction
effort and the assistance of D. Doerfler with the statistical analyses. In addition, the authors thank L. Folinsbee
and L. Birnbaum for their constructive critiques of this manuscript. This manuscript has been reviewed and remains airborne for long periods of time, it
approved for publication by the NHEERL. Mention of trade names or commercial products does not constitute
penetrates the indoor air environment and
endorsement or recommendation for use.
enters the respiratory tract to deposit in the
Address correspondence to Dr. D.L. Costa, Pulmonary Toxicology Branch, MD-82, National Health and
Environmental Effects Research Laboratory; U.S. Environmental Protection Agency, ERC Building, Research airways and deep lung.
Triangle Park, NC 27711. Telephone: (919) 541-2531. Fax: (919) 541-0026. E-mail: costa.dan@epamail.epa.gov
Fly ash from fossil and waste fuel
Abbreviations used: BAL, bronchoalveolar lavage; CFA, coal fly ash; DC, Washington; DOFA, fly ash from a
combustion contributes > 2.5 x 105 tons
domestic oil-burning furnace; Dus, Dusseldorf, Germany; IT, intratracheal; LDH, lactate dehydrogenase; MCT,
monocrotaline; NIST, National Institute of Standards and Technology; Ott, Ottawa, Ontario, Canada; PM, partic- annually to the U.S. ambient PM burden
ulate matter; PM10, particulate matter< 10pm in aerodynamic diameter; PMN, polymorphonuclear neutrophil;
(11). Fly ash is concentrated mostly in
ROFA, residual oil fly ash; St.L, St. Louis, MO; TSP, total suspended particulates; U.S. EPA, U.S. Environmental
urban areas and can be considered a fine
Protection Agency.

Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997 l1053

COSTA AND DREHER

it is likely that any losses or changes in PM
0600- to 1800-hr light cycle. Purina rat
mode PM -that is relatively rich in metal
chemistry due to inherent sample instabil-
chow (Purina Mills, Richmond, IN) and
contaminants (12). To address the hypoth-
ity would have already taken place, even if
water were available ad libitum. Study
esis that PM-related health effects arise
group sizes varied. the sample was subsequently stored under
from anthropogenic combustion-related
controlled conditions designed for preser-
PM with bioavailable transition metals, we
Model Particulate Matter vation. In this sense, the samples used here
elected to use representative emission-
Three types of fly ash were used as emission- must also be considered less than ideal.
source PM. The emission-source fly ash
source particulates to model the combustion They were not of similar age, nor were
selected for study derives from the combus-
component of fine mode PM. Residual oil fly they stored under similar conditions.
tion of domestic and industrial grade fuel
ash (ROFA) was collected as a fugitive ash Their age ranged from about 15 years
oil and coal, and represents a range of
downstream ofan air-cleaning cyclone from a (St.L, DC, Dus) to 3 years (Ott), and they
bioavailable constitutive metals. An isomet-
power plant burning industrial grade #6 low- were either stored frozen (DC, St.L) or at
ric approach was used (equal dose-mass or
S residual oil (13). Fly ash from a domestic room temperature (Dus, Ott) in sealed
equal dose of metal) in an attempt to char-
oil-burning furnace (DOFA) that provided bottles. However, each sample was sieved
acterize the constituents from these sources,
heat for a large apartment complex was col- through a 30-100 mesh to remove large
which are important determinants of the
lected as distal flue-deposited ash. It may be extraneous debris. As such, though there
inflammatory response. In addition, we
considered a mix of both fugitive and settled were likely to be differences in PM organic
used several collected TSP samples to
emission PM, and was provided by A. Ghio character, we felt that the metal content
ascertain the involvement of metals in
of the U.S. Environmental Protection would be relatively stable for the purpose
ambient PM-induced acute lung injury.
Agency (U.S. EPA). The coal fly ash ([CFA] of hypothesis testing. Indeed, comparisons
Previous work with various types of
#4213.3) was collected by the Energy of the metal content of the St.L (SRM
PM suggested that ionizable metals in
Assessment and Control Division, National #1648) and DC (SRM # 1649) dust in
PM correlate with indicators of inflamma-
Exposure Research Laboratory, U.S. EPA, our laboratory with that published by the
tion, altered host resistance, macrophage
from an experimental boiler furnace at the NIST indicated no change. Similarly, the
dysfunction, and airway hyperreactivity
Research Triangle Park, NC research facility. ROFA dust analysis that we conducted for
(13-16), but these studies have not
The source coal was Pittsburgh seam coal, metals (16) was quite similar to that done
attempted to manipulate mass and metal
burned at 825°C using Grove limestone in 1985 (13). We recently showed that
dosing to directly address this concept.
low levels of PM-associated endotoxin did
absorbent (Ca/S ratio = 3/1).
Concomitantly, we established a model of
St. Louis, MO (St.L) and Washington not contribute to the in vivo toxicity of
cardiopulmonary disease in the form of
(DC) PM were purchased from the these ambient PM samples (17).
advanced pulmonary vasculitis/hypertension
The basic physicochemical properties of
National Institute of Standards and
to explore the question of susceptibility
Technology ([NIST] Gaithersburg, MD) each emission and urban PM studied are
and the possible mechanisms by which PM
as SRM #1648 and SRM #1649, respec- provided in Table 1. The samples were pre-
effects are potentiated.
tively. Dusseldorf, Germany (Dus), and pared similarly for characterization of solu-
Methods Ottawa, Ontario, Canada (Ott), PM sam- ble metal and S (presented as SO4) content.
ples were donated by G. Hatch, U.S. EPA, To determine the acid-extractable metal
Animals
and R. Vincent, Health Ministry of content, PM samples were suspended in 1 M
Male Sprague-Dawley rats (60 days of age) Canada, respectively. By their collection HCl in polypropylene tubes at a concentra-
were obtained from Charles River Breeding mode, bagged house TSP samples are less tion of 6.4 to 7.9 mg/ml, followed by mix-
Laboratory (Raleigh, NC) and housed in than ideal. There is little selectivity in the ing end-over-end for 1 to 2 hr at room
an American Association for Assessment of collection process except for the screening temperature. The acid hydrolysates were
Laboratory Animal Care International of relatively large debris and vegetative then centrifuged at 17,000 xg for 20 min,
approved animal facility for at least 1 week fragments, and collection occurs usually then the supernates were transferred to fresh
before use. They were maintained at 22 to over long periods of time with wide-ranging polypropylene tubes for metal analysis.
23°C, 50 to 60% relative humidity, and a ambient humidity and temperature. Thus, Analogously, double-distilled H20 was used

Table 1. Particulate matter physicochemical characterization.
Sizeb/
Total Total
Transition metal contenta
pHc
geometric mean
sulfate
Zn metal
Cu Ni V
Sample Fe
Emission PM
0.12 (100) 358.74 (95)
9.00 (100) 165.70 (86) 1.78/2.02 2.59
2.07 (80)
154.51 (85) 0.00
DOFA
560.52 (100)
103.77 (83) 1.95/2.19 2.99
37.51 (92) 41.71 (84) 1.01 (95)
0.23 (97)
23.31 (68)
ROFA
49.36 (58)
0.37 (0) 0.08 (0) 15.75 (0.4) 4.17/5.00 9.72
0.70 (8.2)
0.03 (0)
14.57 (0)
CFA
Ambient air PM
13.06 (22) 142.66 (93) 3.72/4.42
0.11 (30) 3.56 (73) 5.22
0.11 (35)
0.37 (30)
8.91 (0.4)
StL
137.28 (100)
14.19 (20) 3.58/4.44 5.28
3.70 (68)
0.42 (21) 0.24 (49) 0.18 (31)
9.65 (0.5)
Dus
70.41 (90)
10.63 (57) 18.26(35) 4.09/4.90 6.0
0.27 (30) 0.21 (0.9)
0.82 (18)
6.33 (2.3)
Ott
93.48(91)
1.49(91) 8.42(23) 3.27/3.88 3.65
0.02(100) 0.20(76)
6.57(4.7) 0.14(33)
DC
1 M HCI hydrolysis of PM and expressed in pg/mg PM. Values in parenthesis represent percent of the metal that is water soluble.
quot;Transition metal content obtained from
bMass median aerodynamic diameter of PM. cpH of a 10- to 1 1-mg/ml aqueous suspension.

Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997
1054

METALS IN AMBIENT PM MEDIATE LUNG INJURY

to extract the water-soluble metals, starting samples were centrifuged at 850 xg for metal to that of the mass dose of PM. By
with a concentration of 10 to 11 mg/mi. 1(0 min at 4°C. The recovered cell-free analogy, this approach was applied to
The recovered supernate from this proce- supernates were used for the following bio- ambient urban PM. To this end, three
dure was then acidified to pH < 2.0 with chemical analyses: total protein was deter- emission-source PM, representing a wide
1 M HCI for metal analysis. The concentra- mined using the Coomassie Plus Protein range of varied metal composition and
tions of Fe, V, Ni, Cu, Zn, and SO4= in Assay (Pierce, Rockland, IL); albumin was bioavailability, were evaluated and com-
each extraction were quantified against spe- determined using the MALB SPQ kit pared in the context of four urban TSP
cific standards (Sigma Chemical, St. Louis, (INCSTAR, Stillwater, MN); and lactate from domestic and international sites. The
MO) using a PE Model P40 inductively dehydrogenase (LDH) activity was deter- findings were consistent with other studies,
coupled plasma emission spectrophotometer mined using kit #228 (Sigma Chemical). All which indicated that the toxicity of PM
(Perkin Elmer, Norwalk, CT). PM aerody- assays were adapted for automated analysis generally correlated with total acid-soluble
namic size distributions were determined performed on a Hoffmann-La Roche Cobas metal content, and that the oxidant poten-
using a TSI 3310A aerodynamic particle Fara II dinical analyzer (Roche Diagnostics, tial of the variously solubilized metal
sizer (TSI, St. Paul, MN). The acid-soluble Branchburg, NJ). extracts might be a contributing mecha-
transition metals (Cu, Fe, V, Fe, Zn) com- nism (13-15). The variability in correla-
Animal Model of
mon to all PM were used as the basis for tions both here and in the noted studies
Pulmonary Hypertension
total metal comparisons. may reflect the varied oxidant potential of
An animal model was established to assess transition metals, their multiplicity of
Particulate Maner Exposure the role of preexisting lung disease as a actions, or potential interactions between
and Bronchoalveolar lavage predisposing factor in PM responses. the metals (16).
Each PM was administered by intratracheal Pulmonary vasculitis/hypertension was Table 1 displays the physicochemical
(IT) instillation as previously described by induced in male Sprague Dawley rats 60 characterization of the fly ash and urban PM
Dreher et al. (16). Briefly, a predetermined days of age, using a single ip injection of we examined. Among the fly ash, there was
mass of PM was diluted with sterile, injec- monocrotaline ([MCT] Aldrich Chemical, considerable variation in constituent acid-
tion-grade saline (0.9%) to provide suffi- Milwaukee, WI, 60 mg/kg), as previously soluble metal; the DOFA contained about
cient dosing mixture to dose all the animals described (19). After 10 to 12 days vascu- 50% more total metal than ROFA and
in a group. The rats were anesthetized lar remodeling was well under way, and nearly 10 times that of the CFA. However,
lightly (to lose gagging reflex and response pulmonary arterial pressure was just be while the metal content of ROFA was largely
to pinch) with halothane and instilled IT beginning to increase (19). Pulmonary distributed among Fe, Ni, and V, DOFA-
with 0.3 ml containing the desired PM artery pressure was measured in a limited associated metal was primarily Fe, exceeding
sample, via a transoral tube placed under number of rats (n = 3/group) by catheteriz- that of ROFA by 7- to 10-fold. The Zn con-
laryngoscopic view (18). The animals were ing the pulmonary artery via the jugular tent of DOFA was also relatively high com-
then returned to their cages; they regained vein in the anesthetized intact rat (20). In pared to ROFA, and like much of the
consciousness within approximately 10 a similar cohort of rats, BAL was conducted acid-available metal in oil fly ash, the
min. The treatment doses were based on to ascertain the degree of lung inflamma- majority was water soluble and thus easily
either a) administration of equal dose by tion as described above as well as H2O2 bioavailable. This contrasted with the CFA,
mass (nominal 2.5 mg/rat) of each PM or content (assayed by chemiluminescent which contained a relatively small amount of
b) normalization of each PM mass to a reaction with isoluminol and microperoxi- acid-extractable Fe and even less V, Ni, Cu,
total metal content of 46 pg/dose for the dase). Control and MCT groups (n = 6) and Zn; thus no metal was readily bioavail-
emission PM comparison (see Table 1 for were treated IT with ROFA at doses of 0, able. The pH values for oil fly ash were low
the appropriate metal content) and 35.5 pg 0.25, 1.0, and 2.5 mg/rat. At 96 hr post-IT compared to the CFA, which probably
of total metal (as Cu, Fe, V, Fe, Zn) for the ROFA, the rats underwent BAL as above. reflects the relatively high SO4= content of
ambient PM and ROFA comparison. the former. Oil fly ash is known to associate
Statistl Analysis
At 24 and/or 96 hr following IT instil- with the metals during combustion cooling
lation, the rats were anesthetized with Data were analyzed using one- or two-fac- (K Olin, personal communication).
sodium pentobarbital (Nembutal, Abbott tor analysis of variance and assessed for The ambient PM samples represented
Laboratories, Chicago, IL) (50 mg/kg body overall differences. Pairwise testing (t test) in Table 1 contained about 10% of the
weight, ip), then exsanguinated via the was used, with adjustment for multiple acid-available metal of the oil fly ash, of
abdominal aorta. Rat tracheas were cannu- comparison. The hypothesis tested was which only a small amount was water solu-
lated and bronchoalveolar lavage (BAL) that each PM sample would differ from ble. Therefore, fly ash appears to be a rea-
performed with phosphate-buffered saline control and that specifically for metal- sonable, if significantly more potent,
(Mg+2- and Ca+2-free) pH 7, using a vol- based comparison the PM samples would surrogate for the study of PM mechanisms.
ume equivalent to 28 ml/kg body weight, not differ among themselves. Significance However, it should be noted that because
infused and withdrawn three times. was assigned when the p value was < 0.05. TSP comprises nearly the entire size range
BAL cell counts were determined using a of ambient PM, the indicated metal
Results and Discussion
Coulter Counter (Coulter, Miami, FL). Cell bioavailablity likely underrepresents the
differential analyses were performed on BAL relatively metal-rich respirable fine mode
Metal-based Particulate
cytospin samples prepared using a Shandon PM that would be of most health concern
Matter Toxicity
model cytospin 3 (Shandon Instruments, (19). With regard to bioavailable metal,
Pittsburgh, PA) and stained with LeuKoStat This study attempted to evaluate further the TSP may be considered a mass-diluted
(Fisher Chemical, Pittsburgh, PA). BAL the relative importance of bioavailable form of fine mode PM. In PM S04=, the

Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997 1055

COSTA AND DREHER

*difference was less dramatic (< 50%) than with DOFA, ROFA, and CFA when positive control and, using the extensive
that of oil fly ash. This probably reflects administered in an equimass manner. database developed in this laboratory, fur-
the contributions of other sources of S04= To address the role of metals as the pri- ther insights could be developed. The
such as that from photochemical trans- mary determinant of injury, rats were equimass dosing with urban PM samples
formation of SO2 and perhaps stabilized by treated with each emission fly ash on an and ROFA clearly demonstrated the relative
NH4+ (21). This would account for the equimetal basis (- 46 pg per rat); mass dose potency of fly ash compared to urban PM
lower acidity of TSP relative to that of oil varied. As predicted, the initial injuries samples. With some variability in response,
fly ash. induced by these emission PMs did not all four of the urban PM samples displayed
A wide range of metals was represented about 10% of the ROFA activity in terms of
differ appreciably from each other at the
in the ambient TSP samples, but we 24-hr time point (Figure ID-F). However, BAL injury markers. On the other hand,
elected to focus on the predominant five by 96 hr, some significant differences when all the PMs including ROFA were
that also were predominant in fly ash. The became apparent. In DOFA and CFA, administered in equimetal doses, the magni-
particle size of the ambient TSP samples where the predominant metal was Fe, the tude of the change in the BAL parameters
likely does not represent that which exists equimetal comparison shows similar was similar. Closer scrutiny of the data
responses for all BAL inflammatory para-
in the atmosphere but rather is the product reveals that the Ott PM induces a small but
of agglomerations that occurred during meters at 24 and 96 hr. In contrast, the consistently greater response for all inflam-
collection. However, their size and relative ROFA effect on the BAL parameters at 96 matory markers. Lacking an obvious expla-
metal insolubility compared to oil fly ash hr persisted, which suggested either that nation, one might argue that the excess
prompted us to include the CFA to make one of the metals not in the other fly ash response relates to the somewhat higher
comparisons on the basis of dose mass of had a sustained impact (perhaps Ni) (16) content of water-soluble metal in this PM
the PM challenge. or that metal mixture interactions in the (Table 1) or perhaps a proinflammatory role
Figure IA to C illustrates the impact equimetal dose of ROFA prolonged or of Zn, as suggested by Gavett et al. (22).
of IT-instilled fly ash on BAL parameters altered the longevity of the response, as Alternatively, the Ott was also the freshest
of altered permeability (total protein, reported previously (16). Thus, caution is (most recently collected) PM of the group
albumin) and cellular injury (LDH) at 24 necessary in interpreting bioavailable studied, although a loss of metal potency
and 96 hr after administration. The instil- metal effects when multiple metals may be with age of the PM seems unlikely. The
lation of saline had no significant effect involved. However, a primary role of the potency of ROFA in this study is virtually
on these parameters when compared to bioavailable transition metals in the identical to that previously tested (13).
caged controls (K Dreher, unpublished magnitude of the responses is clear. As with oil fly ash, there was an
data); similarly, acidic instillates have no The impact of fly ash on BAL cellular appreciable eosinophil response associated
effect on these parameters (16). With an profiles (Figure lA-C) when administered with the urban PM samples (Figure 2F).
instillate dose of approximately 2.5 mg fly in accordance with the equimass protocol What is striking is that even with the dilu-
ash, DOFA had the greatest effect, with yielded results in general agreement with tion effect of coarse mode PM mass on
an acute (24 hr) and dramatic alteration the BAL injury parameters. With the that of the fine mode within TSP, the
in permeability and clear evidence of cel- metal-rich oil fly ash, the macrophage and response persists. If one assumes that
lular injury (Figure 1D-F). By 96 hr, per- polymorphonuclear neutrophil (PMN) eosinophilia in BAL is a marker of metal
meability alterations reversed considerably, influxes approached an order of magnitude associated with fly ash as noted above,
although the indicators of cell injury higher cell number when compared to the then this cell type possibly could serve as a
remained high. ROFA also induced con- CFA, which had only a small amount of potential marker of fine mode PM expo-
siderable injury but clearly less than that of bioavailable metal. When administered on sure. Recent studies (K Dreher, unpub-
an equimetal basis (Figure ID-F), the
DOFA; their relative patterns of reversal, lished data) utilizing selectively collected
however, were similar. The degree of inflammatory cell responses are surprisingly fine mode urban PM appear to support
response exceeded that predicted solely on similar in spite of minor fluctuations the contention that eosinophils can serve
that are common to HAL cell data. The
the basis of total metal. As DOFA's metal as such a marker. Likewise, the equimetal
eosinophils, normally considered in the
mix (a ratio of 75:1:17 for Fe:Cu:Zn) dif- studies demonstrated the remarkable con-
fered from that of ROFA (1:1.5:2 for context of allergic or parasitic inflamma- sistency of eosinophilia across ambient PM
samples as well as with ROFA itself, which
Fe:Ni:V) and as there are known interac- tion, respond to the instilled PM and simi-
tions among some transition metals (16), larly appear to be metal mediated. This lends further support to the notion that
varied mechanisms may have been operant. eosinophil response to metals is in many combustion source metals in PM play a
Additionally, it appears that Zn may act ways a curiosity that is characteristic of contributory role in the biologic responses
differently (alone or interactively) in high metal exposure in the context of the PM to ambient PM.
concentrations, as shown in another fly response, and suggests a means of indicting In conclusion, it appears that the
ash that also contained V and Ni, albeit in metal-based activity and anthropogenic induction of injury by emission and
lower concentrations (22). When com- combustion sources in the assessment of ambient PM samples is determined pri-
pared to oil fly ash samples, CFA with ambient PM. marily by constituent metals and their
most of its metal tightly bound induced bioavailability. Based on studies with fly
Examination of Figure 2A to F reveals
that the inflammatory responses to the
little, if any, injury, and none of its para- ash, the earliest phases of the response
meters differed from those of the saline urban PM are coherent with the oil fly ash appear to be driven by the individual met-
control. Clearly, the differential bioavail- samples when linked by the hypothesis that als (16), but the persistence of the
ability of metal content was reflected in the response perhaps reflects the complexity
bioavailable metals drive the response.
dramatically different responses obtained Therefore, ROFA studies could serve as the of the bioavailable metal mix or the

1 056


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83 C5
CS
iF
xRF
0
0
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.SAL
ROFA
SAL DOFA ROFA CFA SAL DOFA CFA DOFA SOFA CFA DOFA
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.

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CRA SAL
DOFA DOFA CFA
SAL ROFA ROFA CFA SAL DOFA ROFA SAL DOFA ROFA CFA
Figure 1. Emission source PM-induced acute pulmonary injury. Results were obtained from analyses performed on BAL samples at 24 and 96 hr postexposure. Rats were
exposed by IT instillation to saline (SAL), ROFA, DOFA, or CFA. Mass refers to responses obtained from animals exposed to an equivalent mass (-2.5 mg/rat) of each PM.
Metal refers to responses obtained from animals exposed to an equivalent amount of total transition metal of each PM (46 pg/rat). This dose of metal translates to the fol-
lowing mass dose of PM: ROFA, 0.450 mg/rat; DOFA, 0.282 mg/rat; and CFA, 2.97 mg/rat. Values are expressed as means +SEM. Animals per group, n=3 to 6 at 24 and 96 hr
postexposure. *, significant difference from saline control. *, significant difference from the other emission samples, excluding SAL.

unique qualities of one metal compared to described herein involve complex mixtures both temporal and dosimetric relationships
another. How the metals are solubilized of metals and other PM-associated com- in need of examination.
in vivo and interact to induce toxicity pounds (i.e., small molecular weight or
Animal Model of
requires further investigation, as redox acidic water-soluble organics) for which
Pulmonary Hypertension
potential is not the same for all the transi- further study is also needed. Clearly, these
tion metals and may depend in part on its conclusions need to be confirmed using MCT is metabolized in the liver to the
coordination state (23). The PM challenges actual inhalation exposures, which involves pyrrole that is specifically toxic to the

Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997 1 057

COSTA AND DREHER

Mass r
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Sal Dus StL DC
Sal Dus StL DC Ott ROFA
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fomfnlye perore do samplsti atW 24or postexposuenc states expoed
toxcit
Thigue2 hasmbienti epM-indued acut pulmonayel jry Results werte obtainceent
inurba were
o mg/rat)lingchPMletalrefes tor
e
by ITistilltioohepahoensistD,Ot OA assrfrtof responstles RObtAie fromanimals texpossbedoa eqiaethu maable f2
pumo
orf
t L s
specfirpsoses). ahP raolw:Ds
talranstinmentatio cPmetal-cpgat- Thedo
e
Typimalsy exposelesion equvaen invouvmnt of
respertnsesotionedfo of
saple,th- uinSL
alyzednifidifrnce uromhe other mbentonM
a
dant
frlomnalin contrtesol;
posresse tosgiicntdiferne t

Fe+3+e-- -oFe+2
by 10 to 12 days, worsens to cause right way. This is due to the substrate-rich
cardiomegaly, then cor pulmonale and milieu of the lung lining fluid during Fe+2 + H202 <- OHquot; + Fe+3
death over the ensuing 3 to 4 weeks (19). inflammation and the weakened state of
Our choice of the model was to establish a the blood-air barrier. The prototypic
preexistent state of progressive inflamma- Fen'ton reaction is depicted below for Fe In vitro assessment of the feasibility of this
tory cardiopulmonary disease as a potential but can be considered applicable to most reaction with ROFA has been positive

1 058


Table 2. Lung inflammation in the monocrotaline cardiopulmonary disease model after 10 to 12 days. 400 SAL rat model
_ MCT rat model b,e
Monocrotaline
Control
BAL end points 350-
b,d
-E
3951.8 ± 423.5*
188.3 ± 30.0
Protein, pg/ml b.c
300
423.5 ± 33.5*
28.3 ± 9.7
LDH, U/liter
462.8 ± 120.0*
PMNs/ml x 103 45.9 ± 0.39 x 250
0.01 75 ± 0.002*
H202, V/0.4x 106 cells 0.0063 ± 0.0011
m
*p<0.01 compared to control.

ash, which has virtually no bioavailable metal
(14,25), but the plausibility of this reaction b
O 100
or significant toxicity in healthy animals, also
in the in vivo model with PM has not been a-
was innocuous in the MCT animal. Mt. St.
verified to date. Theoretically, lung inflam- a,d a,s
as-
so '

Helens volcanic ash was collected for the
mation can result in the release of the reduc- 0
U.S. EPA by A McFarland, Texas A&M
tant superoxide and labile H202 from lung 2.5mg
0.25 mg 1.0mg
0mg
University, from an open field near Ritzville,
cells, which in the presence of lipids can ROFA Dose, mg
WA 340 km NE of the volcano. Thus the
react in vitro with ROFA to yield more oxi-
Figure 3. Neutrophilic response in the BAL fluid in a
PM-associated metal component seems to be
dants (D Costa, unpublished data). More
rat model of pulmonary vasculitis/hypertension
an essential factor for amplification.
work is needed to assess this hypothesis in
induced by a single ip injection of MCT 96 hr after IT
The cause of death in the MCT-ROFA
light of other pathophysiologic frailties of instillation of various doses of ROFA in 0.3 ml saline.
animals appears related to altered cardiac
the impaired animal. Potentiation of the response occurs at each dose of
0

function, although it is unclear whether
Over the 10- to 14-day incubation ROFA; these animals represent the survivor cohort
death was associated with direct cardiac
period after MCT injection, the rats lost (-50%) at the time of analysis. Like-lettered super-
injury or was secondary to pulmonary fail-
little body weight (- 10%); however, pul- scripts indicate significantly different from the appro-
ure (26). Electrocardiographic study
monary artery pressure measured in a small priate control or ROFA alone (- MCT) group.
revealed that ROFA alone in the healthy
cohort of similarly treated rats indicated a
rat clearly induced a mild arrhythmic con-
doubling of Ppa from 10 to 20 mm Hg. Table 3. Mortality of monocrotaline rat model treated
dition over the acute inflammatory time
Baseline BAL values in Table 2 for control with intratracheal residual oil fly ash.
period (96 hr). However, animals pre-
and MCT rats reflect the inflammatory
Mortality by 96 hr
treated with MCT exhibited significantly Treatment
state of the MCT model at the point of IT
more and worsened dysrhythmias, among
instillation of ROFA. 4/46
MCT + saline
which bundle branch A-V block and S-T
Ninety-six hours after ROFA instillation, MCT + 0.25 mg ROFA 8/16
segment inversion were considered most 10/16
there was clear evidence of enhanced neu- MCT + 1.0 mg ROFA
25/46
MCT + 2.5 mg ROFA
severe. These alterations progressed with
trophilic inflammation (Figure 3), with the
time and correlated with time of death.
influx of PMNs appearing to plateau at 1
Though their origin is as yet uncertain,
mg ROFA/rat. However, other BAL end
healthy and the at-risk compromised host.
these cardiographic changes appear consis-
points did not show a consistent picture of
When adjusted for bioavailable metal con-
tent with the formation of ectopic foci in
enhanced injury because of great variability
tent, combustion source and ambient PM
heart muscle and/or hypoxic ischemia, pos-
in response (data not shown). Notably, by
(TSP) show remarkable similarity in the
sibly secondary to pulmonary diffusion
96 hr, significant mortality had occurred in
inflammatory response. The data in the
abnormalities (S Gardner, unpublished
the MCT-ROFA groups (050% across all
compromised animals appear to parallel the
data). This model has not yet been utilized
doses of ROFA), with the remaining animals
recent epidemiology studies suggesting that
with ambient PM in this laboratory; how-
in a somewhat more impaired state than
those at enhanced risk possess chronic
ever, Godleski et al. (27) reported mortal-
either MCT or ROFA alone. Thus, interpre-
inflammatory lung conditions. The current
ity in a similar model with inhalation of
tation of any 96-hr data is representative of
need is to ascertain the relevance of these
concentrated ambient Boston, MA air
the survivor response. Table 3 sums our
(-350 pg/M3 for 6 hr/day for 3 days). data and validate the hypothesis that metals
experience with the MCT model regarding
mediate toxicity when applied to inhalation
mortality. To date, there does not appear to
Conclusion exposure scenarios that approximate similar
be a dose response over the range of doses
doses in humans.
We propose that soluble metals from PM
evaluated; time to death also appears not to
mediate the array of PM-associated injuries
be dose dependent. Preliminary studies in
to the cardiopulmonary system of both the
our laboratory with Mt. St. Helens volcanic

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