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OCR for page 97
Given the high mutagenic potency of nitro-PAHs, it appears appropriate
to speculate on the fate of these compounds in ambient air. Four
nit r~-PAHs have been reported in urban particulate matter--6-nitro-
BaP, 1 3-nitrofluoranthene, 1, 5 1-nitropyrene, and 5-nitroacenaph-
thene85--and indirect evidence of the presence of nitro-PAHs in Wayne
County3 Michigan, air has been presented on the basis of mutagenicity
assays conducted with nitroreductase-deficient strains.483 89 Photolysis
of nitro-PAHs, such as 9-nitroanthracene, yields the corresponding diones
(e.g., 9,10-anthraquinone), both in solutig29 and on silica gel.7 On
exposure of pyrene to N02' Jager and Hanus noted the appearance of
new products after 4 h, and the nitropyrene yield decreased substan-
tially. However, the retention times of these as yet unidentified com-
pounds were different from those of the pyrene diones. The atmospheric
relevance of these and other pathways should be investigated further.
REACTIONS OF PAHs WITH SULFUR DIOXIDE
Jager and Rakovic43 have reported the fonnation of sulfonic acids
and other sulfur-containing compounds on exposure of BaP and pyrene
(adsorbed on fly ash and on alumina) to SO2 at a high concentration (10%
in air). These sulfonic acids are also easily prepared in the liquid
phase by reaction of PAHs with sulfuric acid at room temperature.88
Tebbens et al.82 observed significant consumption of BaP adsorbed on
soot from a propane burner and exposed to SO2 at 50-80 ppm for 4 h, both
in the dark and under irradiation.
At lower SO2 concentrations more relevant to ambient pollution, PAHs
do not appear to react readily with SO2. Hughes et al.37 observed no
reaction between SO2 at parts-per-million concentrations and BaP or
pyrene adsorbed on silica gel, alumina, and coal fly ash. Butler and
Crossley7 exposed 20 PAHs adsorbed on carbon (soot) to SO2 at 5 ppm in
air for up to 100 d. The matrix air contained water vapor, and thus some
sulfuric acid was presumably present. Within the stated analytic preci-
sion, no significant reaction was observed for phenanthrene, coronene,
fluoranthene, chrysene, BaP, pyrene, benz~ajanthracene, benzotghi]-
perylene, and anthanthrene. Because the 20 PAHs studied include both
highly reactive and essentially inert compounds with respect to reaction
with, for example, O3 and N02, the conclusions of Butler and
Crossley7 as to the absence of significant reaction with SO2 can
probably be extended to many of the PAHs present in polluted air.
However, SO2 and sulfuric acid may play a role as catalysts for other
PAH reactions, including nitration, and this possible catalytic role
should be inves ligated.
REACTIONS OF PAHs WITH OTHER OXIDIZING SPECIES
Reactions of adsorbed PAHs with atmospheric pollutants other than
03, S02, and NO2 have received very little attention. Pitts et
_ .75 exposed BaP deposited on a glass filter to peroxyacetylnitrate
3-11
OCR for page 98
[CH3CO(OO)NO2, or PAN] at about 1 ppm for 16 h and observed the
formation of BaP diones and other oxidation products. Ambient concen-
trations of PAN in the Los Angeles atmosphere often reach 30 ppb during
episodes of photochemical smog,29 so PAN may contribute, with Of, to
the oxidative degradation of PAHs in photochemically polluted air.
Reactions of PAHs with free radicals, including the hydroxyl (OH) and
hydroperoxyl (HO2) radicals (well documented in the bulk liquid phase),
have not been studied in the context of atmospheric pollution. On the
basis of studies conducted with aromatic compounds, such as toluene, the
OH photooxidation products in the presence of NOx include particulate-
phase hydroxynitrotoluene and dihydroxynitrotoluene as major pro-
ducts.31 It is possible that atmospheric oxidation of PAHs initiated by
reaction with the OH radical results in the formation of nitro, hydroxy,
and hydroxynitro derivatives.
PHOTOCHEMICAL REACT IONS OF PAHs
The mechanisms involved in photochemical reactions of PAHs with
singlet oxygen in the bulk-liquid phase have received considerable atten-
tion and have been the object of several comprehensive reviews.24~66 As
is illustrated in Figure 3-6 for anthracene, products of these reactions
include the PAR diones, formed either directly or by further reaction of
primary endoperoxide products, as well as other oxygenated compounds.
Photomodifications of BaP and other PAHs in the adsorbed state have
received significant attention with respect to both product distribu-
tion and influence of substrate. Product studies are in good agree-
ment, and the chemical distribution of PAR phototransformations in the
adsorbed state closely resembles that obtained in the bulk-liquid phase.
However, reactivity reportedly varies widely as a function of substrate,
and that makes it difficult to extrapolate laboratory studies to the
ambient atmosphere.
Falk, Markul, and Kotinl7 first reported on the photodecomposi-
tion of 10 PAHs, including BaP, deposited on Whatman #1 paper filters and
exposed to air in the dark, to air under irradiation, and to synthetic
smog prepared by the reaction of O3 with gasoline. Their experiments
were conducted with pure PAHs, as well as with PAHs adsorbed on
gasoline-engine exhaust soot. On exposure of light in air, all PAHs
adsorbed on soot were more resistant to photodecomposition than the same
compounds in the pure form. Tebbens et al.81~82 investigated photo-
transformations of BaP, perylene' pyrene, and fluoranthene adsorbed on
soot or deposited on paper, acetylated, and glass-fiber filters. Losses
of BaP of up to 40: were observed on irradiation for some 45 min in air;
the major reaction products were the three BaP diones and a carboxylic
acid derivative. Thomas _ al.83 reported similar results for BaP.
Phototransformations of BaP and other PAHs have also been observed on
a variety of substrates, including alumina,39~51 silica gel,39~40~5
cellulose,39~45 acetylated cellulose,39 soil,l9 carbon micro
3-12
OCR for page 99
needles,3 atmospheric particulate matter,21 and coal fly ash.50 A
summary of the products of heterogeneous photooxidation of BaP on various
substrates is given in Figure 3-7.
For comparison, Table 3-8 lists half-lives and percent losses
cellulose TLC plates45 and
determined for a number of PAHs deposited on
on Whatman #1 paperl7 and adsorbed on sootl7 and on fly ash.50
Although the four sets of data are directly comparable for only two
compounds, perylene and BaP, the effect of substrate on PAH phototrans-
formations is evident. PAHs deposited in the pure form on cellulose TLC
plates exhibit short half-lives, from 23 min for anthracene to about 20 h
for BaP.45 On Whatman paper, pure PAHs appear more resistant to
photooxidation; egg., the half-life for perylene is about 2 d, compared
with only 4 h on cellulose TLC plates. In the adsorbed state, PAHs appear
to be much more resistant to photooxidation, with losses of only 10Z on
soot after 48 h of irradiation.17 On fly ash, only modest photodecom-
position rates (up to 20%) are observed, in striking contrast with rapid
photooxidation in the liquid phase and on silica gel.50 If one neglects
in a first approximation the important differences in experimental
conditions, it appears from the data listed in Table 3-8 that PAHs
adsorbed on atmospheric particles may be somewhat resistant to photo-
oxidation, with half-lives ranging from several days to several weeks,
depending on the reactivity of each compound.
Korfmacher _ al.50 have discussed the possible physical and
chemical factors involved in the resistance of PAHs to-photooxidation when
adsorbed on fly ash. Resistance to photooxidation on soot, although even
more relevant to urban pollution, where submicrometer particles contain a
substantial fraction of carbonaceous material,30 has not been fully
investigated. In addition, specific PAH-substrate interactions have to be
considered. For example, Korfmacher et al.49 and Kotin et al.53
observed rapid decomposition in the dark of some PAHs adsorbed on fly ash
and on soot.
Until more data become available, caution must be exercised in
extrapolating laboratory results to PAH photooxidation in the atmosphere.
INTERACTIONS OF DEPOSITED PAHs WITH AMBIENT POLLUTANTS
It i8 somewhat surprising 3 in view of the critical need to obtain
overall PAH chemical deposition rates over a range of ambient condi-
tions, that only a few studies have investigated interactions of PAHs with
ambient polluted air. Pitts _ al.75 exposed pure BaP deposited on a
glass filter to particle-free ambient Riverside, California, air for 3 d.
BaP was partially oxidized under these conditions, yielding BaP diones and
a variety of oxygenated (but not nitro) derivatives. In contrast, Fox and
Olive21 found only trace amounts of anthraquinone from anthracene (a
reactive PAH in the studies discussed above) adsorbed on ambient parti-
culate matter (suburban location near Austin, Texas) and exposed to atmos-
pheric gases for 4 d in the dark. Comparison of the results of these two
studies suggests that, as noted for photooxidation, PAHs appear more
3-13
OCR for page 100
resistant to degradation in the adsorbed state than in the pure form.
Peters and Seifert70 exposed glass-f iber filters impregnated with
4C-labeled BaP to ambient air in Berlin, Germany, and noted substantial
losses of BaP, typically 75% over 24 h. Simultaneous determination of
14C activity 'only 10% loss in 24 h) established that BaP losses were
due to chemical reaction, rather than to BaP evaporation from the filter.
In addition, a relationship was noted between BaP reaction rates and
ambient O3 concentrations.
A recent investigation of PAH concentrations in the plume of a
coal-fired power plant as a function of distance from the stack has been
reported by Kalkwarf and Garcia. 44 Fluoranthene ~ BaP ~ pyrene ~ and BeP
in the plume were found to be 50% reacted 3, 6, 8, and 12 kn from the
stack, respectively (with correction for plume dilution and dispersion).
The loss of the PAHs was attributed to their reaction with committed NO2
and SO2.
PAHs IN AMBIENT AIR
Source identification i s a key problem in the development of a
pollution abatement or control program. In 1973, Friedlander22 des-
cribed a technique to identify the sources of air pollutants in emission
inventories for particulate matter. Many chemical elements--such as
sodium, chlorine, silicon, and aluminum--are found in natural back-
ground aerosols of the atmospheres of urban and industrial basins, such as
Los Angeles. These are differentiated from other chemical tracers--such
as lead, vanadium, zinc, and barium--which are attributable to human
activities ~ see Figure 3-8) . Thus, if some of the ma jor sources are known
in a given area, the source contributors to the atmosphere can be iden-
tif fed and calculated by measuring the elemental concentrations at a given
point and f itting the data into a mathematical model. One of the ma jar
problems in using this technique has been the need for instrumentation for
real-time measurement of the tracer elements. The use of trace metals for
identification of sources of particles was examined by Moyers et al. in
1977.65 With these tracers, several sources of particulate species in
desert, rural, and urban atmospheres could be determined.
In 1979, Daisey et al.l4 described three methods for source identi-
fication for the PAHs in the complex mix of the atmosphere. Although the
evaluations of these methods are in the early stages, it was found that
statistical modeling does not depend on source emission data, if the
ambient-air measurement data base is large. In 1981, Daisey and Kneipl3
reported that it was possible to use multivariate regression models of
ambient-air data for apportioning the contributions of emission sources to
airborne particulate organic matter. The contributing sources of respir-
able particles were determined by analysis of the ambient-air measurement
data taken in New York City: 19: were from automobiles and related
sources, 40Z were from oil-burning, and 15% were soil-like particles.
Although this study using tracer chemicals had good results, the methods
should be validated for predictive use by testing in other locations.
3-14
OCR for page 101
A comprehensive discussion and critique of environmental sampling and
analytic methods used for polycyclic organic matter are in the EPA
report.79 Lee et al. ,58 in a book on the analytic chemistry of PAHs,
discussed sampling of mobile and stationary sources, ambient air, water,
food, soils, and the aquatic environment. The cleanup and separation pro-
cesses for the various collection media include solvent partitioning for
analysis by column, paper, thin-layer, gas, and high-pressure liquid
chromatography. The percentages of recovery with the analytic methods for
the various PAHs were described by Lee et al., but are not discussed here.
In 1967, Hangebrauck et al.,34 in a review of known sources of PAHs,
gave the results of a survey made by the National Center for Air Pollution
Control to screen the processes likely to produce emission in urban air.
Although the survey was not intended to establish statistically the
average emission from the sources, the data from it do characterize and
classify the rate of emission of several PAHs from four major source
categories: heat generation, refuse-burning, industrial processes, and
motor vehicles. The 1972 NRC report Particulate Polycyclic Organic
Mattered estimated that heat and power generation produced BaP at SOO
metric tons/yr; refuse-burning, at 600 tons/yr; coke production, at 200
tons/yr; and motor vehicles, at 20 tons/yr. BaP has often been used as a
surrogate in estimating source contributions of complex mixtures of PAHs.
Surrogate chemicals have been used commonly in monitoring environ-
mental quality, for various reasons--e."., analytic methods are often
available only for the surrogate, and it costs less to monitor only one
chemical. However, a PAH surrogate may not be useful unless studies have
been conducted to characterize the PAH profile and percentage relation-
shi~s for each type of environment. For example, in 1979, Bjorseth et
al. showed that the relative distribution of PAHs is not the same in
all environments. Figure 3-9 shows comparisons of the percentages of PAHs
found in the particulate matter from an aluminum plant and a Soderberg
paste plant. He recommended that a parent PAH profile (PPP) be estab-
lished before a surrogate compound was chosen. In 1981, Gammage and
Bjorseth23 stated that there are no established techniques for real-time
monitoring of selected PAHs and that BaP is not a universally accepted
proxy or surrogate for PAHs. It is known that the numerous PAHs found in
the outdoor air can be radically different, qualitatively and quanti-
tatively, from those in the workplace environment and that monitoring one
compound as a surrogate for others is unreliable. The recommendation was
made again that a PEP be determined before a proxy or surrogate compound
was chosen.
Cons iderable information is available to show the profile of the
-various PAHs from various sources. Comparison of the rate of PAH emission
between different categories is complicated, owing to the different units
used to measure them: Ug/BTU, ~g/barrel of oil, Agog of particles, ~g/lb
of material processed, ~g/m3, ~g/mi. Qualitative comparisons of PAH
amiss ion sources do appear feasible and can identify the various contri-
butors to the overall pollution burden. Lists of the PAHs found in the
3-15
OCR for page 102
following five broad categories have characterized the variety of sources
and identified some of the major contributors: heat and power generation,
refuse-burning, industrial processes, motor vehicles, and natural sources.
In the 1978 review of the sources of PAHs, Baum,4 using data
assembled by EPA in 1974, estimated that 97% of the BaP emitted in the
United States could be attributed to stationary fuel combustion. The
major contributors were the inefficient combustion of coal in residential
furnaces, coke ovens, and refuse fires. This is in close agreement with
the NRC report Particulate Polycyclic Organic Matter, which stated that
90% of the annual nationwide BaP emission was attributable to coal- and
wood-fired residential furnaces, coal-refuse fires, and coke production.
A wide range in concentrations of BaP (0.1-388 ng/m3) has been
reported by Colucci and Begemanl0 for U.S. and foreign cities (see Table
3-9). These results are for measurements taken between 1952 and 1966. The
objective of the authors was to study BaP concentrations in the atmosphere
in relation to automobile traffic. They used tracers to identify auto-
motive and nonautomotive sources and calculated correlation coefficients
of BaP with CO (a motor-vehicle tracer) as 0.65, with lead (a gasoline-
vehicle tracer) as 0.74, and with vanadium (an oil tracer) as 0.54.*
The seasonal variations show that the concentrations were highest in fall
and winter and lowest in spring and summer. The winter vanadium concen-
trations were twice the summer concentrations; that indicates that the
higher amount of BaP was attributable to combustion of residual fuels used
for heating or to the lower inversion heights prevalent during cold
weather.
The concentrations of BaP in England, Italy, Norway, Sweden, and
GermanOy, as shown in Table 3-10 for 1953-1964, were given in the report by
Louw,6 for the purpose of comparing the findings Sin South Africa. The
concentrations of BaP ranged between 5 and 49 ng/m . One sample, taken
near a road-tarring operation, was extremely high' 1~113 ng/m ~
In Ontario, Canada, five locations were sampled for BaP by Katz et
al.46 from April 1975 through March 1976. The highest concentration was
obse3rved in Hamilton (3,498 ng/m3), and the lowest in Sudbury (111
ng~m ); the latter was attributed to the electrostatic precipitators in
use at the nickel-copper smelter 5 mi away. The concentrations are given
in Table 3-11.
A study was conducted in Karlsruhe, Germany, to determine the relative
amounts of BaP from residential heating systems and automobile traffic
(see Figure 3-10). The concentrations ranged from 0.1 ng/m3 (at the
low-traffic Municipal Garden) to 28 ng/m3 (at a railroad underpass).
With lead as the tracer, it was determined that the highest concentration
caused by automobiles was in the underpass. The low concentration in the
lithe correlations indicate that 42-55% of the variation in BaP concen-
trations is related to motor-vehicle tracers and 29% to stationary
oil-burning.
3-16
OCR for page 103
Municipal Garden during summer was attributed to deposition of airborne
particles on leaves, trees, and shrubs. During the winter, the increase
in the concentration of BaP was attributed to increased residential heat-
ing. The air samples taken at Karlsruhe Nuclear Research Center, 11 km
(by air) north of the city, had the lowest concentrations, except for
those in the Garden during May and June.
BaP was determined in four locations around the industrial city of
Essen, Germany, by Grimmer et al.,28 from October 1978 to March 1979.
There were four sampling sites at each of five locations. The authors
concluded that the concentrations of BaP in ambient air varied by a factor
of more than 10 from one station to another during the cold-weather heat-
ing period. Thus, they did not give any average values; the approximate
ranges of concentration at each location were as follows: 1-75 ng/m3 in
an area that used hand-stoked coal-heating in residences,-1.5-21 ng/m3
in an area with oil-heating only, 10-100 ng/m3 in a tunnel with car
traffic, 15-210 ng/m3 in an area with coke ovens, and 1-75 ng/m3 in an
area described as rural, outside the city.
Two very thorough studies of the PAR content of Los Angeles air have
been made by Gordon and Bryan26 and Gordon.25 The earlier study was
of four locations in the Los Angeles basin (see Figure 3-11), and the
latter included 13 sampling locations ~ see Figure 3-12 ~ . Analyses were
performed for 14 PAHs, including BaP, sampled over the course of a year.
From the relationship between meteorology, traffic density, and PAR
concentrations, the authors concluded that most (at least 607) of the PAHs
was contributed by automobile traffic, but that the concentrations were
lower than in many other cities. This result was expected, because of the
extensive use of natural gas and hydrothermal energy in the West and the
nonuse of coal in Los Angeles. The warm climate also limits wood-burning
in fireplaces. The Colucci and Begemanll results demonstrated much
higher ambient BaP concentrations in urban areas that depend extensively
on coal, oil, and wood combustion. They determined that the automotive
contribution to Detroit ambient BaP was only 5-42%, with typical BaP
concentrations 3 times as high as in Los Angeles.
3-17
OCR for page 104
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OCR for page 134
78. Santodonato, J., P. Howard, D. Basu, S. Lande, J. K. Selkirk, and P.
Sheehe. Health Assessment Document for Polycyclic Organic Matter.
Report EPA-600/9-79-008. Research Triangle Park, N.C.: U.S. Environ-
mental Protection Agency, Office of Health and Environmental Assess-
ment, 1979. [475] pp. (preprint)
79. Santodonato, J., P. Howard, D. Basu, S. D. Lee, and L. Grant, Eds.
Health and Ecological Assessment of Polynuclear Aromatic Hydrocarbons.
Environ. Path. Toxicol. (Special Issue) 5~1~:1-372, 1981.
80. Sehmel, G. A., and S. L. Butter. Particle deposition rates on a water
surface as a function of particle diameter and air velocity. J. Rech.
Atmospheriques 8:911-920, 1974.
81. Tebbens, B. D., M. Mukai, and J. F. Thomas. Fate of arenes incorporated
with airborne soot: Effect of irradiation. Amer. Ind. Hyg. Assoc. J.
32:365-372, 1971.
Tebbens, B. D., J. P. Thomas, and M. Mukai. Fate of arenes incorporated
with airborne soot. Amer. Ind. Hyg. Assoc. J. 27:415-422, 1966.
83. Thomas, J. F., M. Mukai, and B. D. Tebbens. Fate of airborne benzo[a]-
pyrene. Environ. Sci. Technol. 2:33-39, 1968.
84. Tokiwa, H., K. Nakagawa, K. Morita, and Y. Ohnisha. Mutagenicity of
nitro derivatives induced by exposure of aromatic compounds to nitrogen
dioxide. Mutat. Res. 91: 195-205, 1981.
85. Tokiwa, H., R. Nakagawa, and Y. Ohnishi. Mutagenic assay of aromatic
nitro compounds with Salmonella typhimurium. Mutat. Res. 91:321-325,
1981.
86. Van Vaeck, L., G. Broddin, and K. Van Cauwenberghe. On the relevance of
air pollution measurements of aliphatic and polyaromatic hydrocarbons in
ambient particulate matter. Biomed. Mass Spectrom. 7:473-483, 1980.
81. Van Vaeck, L.' and K. Van Cauwenberghe. Cascade impactor measurements of
the size distribution of the major classes of organic pollutants in
atmospheric particulate matter. Atmos. Environ. 12:2229-2239, 1978.
88. Vollmann, H., H. Becker, M. Corell, and H. Streeck. Beitrage zur
Kenntnis des Pyrens und seiner Derivate. Justus Liebigs Ann. Chem. 531:
1-159, 1937.
89. Wang, C. Y., M-S. Lee, C. M. King, and P. 0. Warner. Evidence for nitro-
aromatics as direct-acting mutagens of airborne particles. Chemosphere
9:83-87, 1980.
90. Windsor, J. G., Jr., and R. A. Hites. PolycycLic aromatic hydrocarbons
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Acta 43: 27-33, 1979.
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4
BIOLOGIC EFFECTS OF SMOKE, EMISSION, AND SOME OF THEIR PAH COMPONENTS
The environment is a major contributor to the development of a
variety of pathologic conditions in humans. Indeed, Doll and Peto43
have estimated that as much as 13% of all human deaths from cancer may
be attributed to exposure to harmful polluting substances in our
environment. The purposes of this chapter are to describe the biologic
activity of various kinds of polluting emission and some of their PAH
components and to attempt to relate the toxic impact of such emission
to its content of specific PAHs. (See Chapter 3 for discussion of
particle size and respiratory uptake, Chapter 6 for discussion of PAH
transfer in tissues, and Chapter 9 for recommendations.) It considers
biologic activity in bacteria, animal-cell systems, and intact animals,
as well as the nature and advantages of some biologic models used in
emission toxicology.
Hilado and colleagues65~68 have reported considerable morbidity
in experimental animals that were exposed to the products of combustion
of hard woods, such as birch and oak, or soft woods, such as fir and
pine; they noted no difference in toxicity between the products of
these hard and soft woods. The problem of interpreting results related
to wood is compounded by the presence of preservatives and other
additives in the wood. It is often difficult to establish whether any
observed toxicity is caused by the combustion products of the wood
itself or of a contaminating additive. And it has not been established
whether the PAHs generated during combustion contribute more to the
observed toxicity than the gaseous products. Considerable additional
work with subacute and chronic exposure is required to characterize
toxicity, particularly in view of the current increase in such emission.
Toxicity has been measured in rats and mice intermittently expose'
to diesel exhaust for periods up to 308 h. 110 The total cumulative
particulate exposure varied from 7.75 to 1,310 mg/m3-h. However,
only minimal changes from normal were observed. Glutathione reductase
and lactic acid dehydrogenase activities, which might serve as
indicators of lung-cell damage, were increased in ravage fluid after
wk of exposure at the high dosage; although exposure was continued,
they returned to normal by 6 wk. Neutral protease activity was
increased in ravage fluid after 1 wk of exposure at the medium dosage
(30.6 mg/m3-h) and the high dosage, but returned to normal by the
twelfth week of continued exposure. It is of interest that no
alteration in cytochrome P450 activity was observed in either mouse
or rat liver at any time in any group. After 12 wk of exposure at the
highest dosage, an increase in the number of macrophages was seen in
the ravage fluid.
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tar
TOXICITY TO SPECIFIC ORGANS AND ORGAN SYSTEMS IN ANIMALS
. ..
Manifestations of toxicity to specific organs and organ systems
were detected in animals that were exposed to various kinds of emission.
PULMONARY FUNCT ION
.
Many studies have been conducted in which animals were exposed to
dielel-exhaust particles (generally 0.1-0.2 Am in size). Abraham et
al. reported little change in pulmonary resistance or in airway
reactivity to a carbachol aerosol in conscious sheep exposed for 30 min
to diesel-exhaust particles. Battigelli7 exposed human volunteers
for up to 1 h to diesel exhaust at total hydrocarbon concentrations of
2-6 ppm (comparable with the environment in railroad shops) and then
measured pulmonary resistance as an index of function. With this
rather insensitive assay of only relatively short duration, no changes
in function were observed.
Mauderly et al.ll° measured tracheal mucociliary clearance of a
99mTc-macroaggregated albumin suspension that had been instilled
intratracheally in rats 1 wk before exposure to diesel exhaust for 1,
6, or 12 wk. They also examined the morphology o f the lung and trachea
with scanning electron microscopy. In the group of animals that had
been exposed at high dosages (cumulative particle exposure of 151, 822,
or 1,310 mg/m3-h, respectively, after 1, 6, or 12 wk of exposure),
clearance of the suspension was increased after 1 wk; by 12 wk. it was
below normal. In this group, a tendency toward reduced numbers of
ciliated cells was noted. Furthermore, a dose-related increase in
pulmonary macrophages was apparent. Many of these cells contained
diesel particles as inclusions. No changes were seen in the morphology
of the alveoli or airways. In the groups of animals that were exposed
during the same times at lower dosages (30.6, 203, or 317 mg/m3-h), a
reduction in clearance was the more prevalent response. However, in
the pulmonary function part of this study, in no group of exposed mice
or rats was any significant alteration in pulmonary function observed.
A similar lack of effect on pulmonary function after diesel-exhaust
exposure of rats was reported by Pepelko 5 and by Gross.58 In the
study of Pepelko et al., rats were exposed for 20 in/d, 7 d/wk, for 28 d
to a 1:4 raw or irradiated exhaust from a six-cylinder Nissan diesel
engine. Gross exposed rats to diesel-exhaust particles at 1,500
~g/m3 for 20 in/d, 5 d/wk, for up to 267 d, but suggested that a
longer chronic exposure of the rats to the particles might result in
lung disease.
Because guinea pigs are generally more susceptible to pulmonary
lesions, they were similarly exposed to diesel-exhaust particles for
periods varying from 2 wk to 3 mo at 250-6,000 ~g/m3. As reported
for other species, the number of pulmonary a lveolar macrophages
OCR for page 137
increased, and they tended to accumulate at the bronchoalveolar
junctions. Occasional localization of the particles in alveolar Type
epithelial cells and sporadic increases in Type II cells were
observed. However, all these morphologic changes would be classified
as minimal with regard to pulmonary toxicity.
A different tack was taken by Mauderly et al.ll° and by Campbell
_ al.l7 to demonstrate an alteration in pulmonary function after
exposure of rats or mice to diesel-exhaust particles. In the former
study, exposed rats were inoculated with 32P-labeled Pseudomonas
aeruginosa at the oropharynx, and the killing and clearance of these
organisms were ascertained 48 h later; no significant difference in
either measure was observed. In the latter study, mice that had been
exposed to light-duty diesel exhaust (up to 8 in/d, 7 d/wk, for 46 wk)
were treated with aerosols of Streptococcus pyogenes or Salmonella
typhimurium; mice that had been exposed to exhaust showed slightly
increased toxic responses to streptococci. These results were
confirmed in later studies by Campbell et al.17a showing greater
mortality of infected mice exposed to diesel than to gasoline
(catalyst-treated) engine exhausts.
In brief, minimal changes are observed in pulmonary function and
morphology after exposure to diesel-exhaust particles. Although many
morphologic studies have been conducted in animals that have received
some individual PAHs intratracheally or otherwise, there is little
information on resulting alterations in pulmonary function. The
morphologic changes that are generally classified as metaplastic are
discussed later in this chapter.
NERVOUS SYSTEM
Evaluating the effects of any potential toxin on the development
and function of the nervous system experimentally is very difficult.
Laurie and colleagues97~98 set about to determine the effects of
chronic diesel-exhaust exposure of neonatal rats on spontaneous
locomotor activity and on performance in a bar-pres~ing task. The
neonatal rats were exposed to the exhaust at 6 mg/m for 8-20 hid for
17-42 d, starting on day 1 or 2 of life. Performance was assessed
during weeks 5-16. The activity was depressed both during exposure and
in the group tested after exposure, compared with a control group;
i.e., they required more extensive training. Because published reports
had indicated that the gaseous components lacked any such effect, the
authors concluded that the particles or their PAH components were the
responsible factors. Laurie and Boyes97 measured the somatosensory
and visual evoked potentials in control rats and rats that had been
exposed to diesel exhaust during neonatal life. Although only small
abnormalities were noted in the visual evoked potential, significantly
longer latencies for all the peaks of somatosensory evoked potential
were seen in the exposed rats. Because the latter potentials are in
the central nervous system, the authors suggested that diesel-exhaust
exposure may lead to failure to develop a normally functioning nervous
system. These types of studies have not been conducted with animals
exposed to individual PAHs, or to mixtures thereof, so it is not known
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whether these organic components are responsible for the nervous system
lesions.
IMMUNE SYSTEM
.
The effects of chronic diesel-exhaust exposure of rats on the
immune system were assessed by Mauderly et al.ll° They placed rats
in chambers and exposed them to diesel-exhaust particles for various
periods under dynamic conditions and gave them sheep red blood cells
intratracheally. The numbers of lymphoid cells that produced IgM
antibody to sheep red cells were determined in lymph nodes and spleen 7
d after inoculation with the sheep red cells. Only minimal or no
effects on the induction of immunity were observed.
Those studies are of particular interest, in view of the long-
known damaging effect of some PAHs on lymphoid tissue. In 1937, Haddow
and co-workers59 reported the systemic toxic effects of PAHs, calling
attention particularly to damage to lymphoid tissue. Acute exposure of
mice to 3-methylcholanthrene reportedly resulted in damage to the
thymus that was followed by thymoma formation, a marked reduction in
the weights of the spleen and the mesenteric lymph nodes, and
degeneration of bone marrow cells.l84 Newborns appeared particularly
sensitive, suffering a wasting disease that culminated in death. This
toxic effect has also been noted after administration of
7,12-dimethylbenzanthracene (7,12-DMBA) to rats.23~128 Repeated
administrations of dibenz[ah]anthracene, benz[a]anthracene, or anthra-
cene to mice resulted in an increase in stem cells in lymph glands, a
decrease in mature lymphoid cells and a decrease in spleen weight
(only for dibenz[ah]anthracene).76 In rats, findings were similar
after treatment with dibenz[ah~anthracene; the effects with anthracene
were much less dramatic.
The total immune response of an organism is an expression of the
sum of humoral and cell-mediated effects. Humoral effects derive from
the activity of B lymphocytes, which on maturation to plasma cells
elaborate immunoglobulins; cell-mediated immunity is expressed by T
cells. The effects of chronic administration of benzo[a]pyrene (daily
subcutaneous injection for 14 d, for a total of 50-400 mg/kg of body
weight) on the humoral immune response were summarized by Dean et
al. 8 There was a marked decrease in this response. (The
noncarcinogen benzo[e]pyrene (BeP) was without effect.) A variety of
T-cell responses have been tested for sensitivity to benzotaipyrene
(BaP) administration.l2~38~39,l04 The effects of chronic BaP
administration, to a total of 400 mg/kg of body weight' on T-cell
function were much less marked than those on B-cell activity. Little
effect on the incidence of the B6 tumor in inoculated mice or on the
growth of the B16 melanoma after intravenous challenge was observed.
Furthermore, the resistance of mice to Listeria monocytogenes was
unaltered by administration of BaP, although the expulsion of the
parasite Trichinella spiralis was reduced. BaP administration resulted
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in myelotoxicity, as determined by in vitro clonal bone-marrow assays.
But delayed hypersensitivity reactions in the host were unaffected.
summary, the effects of BaP administration on a variety of T-cell
functions were not very s igni ficant.
It has long been known that care inogenic PAHs are i~mnunosuppres-
sive; indeed, this aspect of their action was believed responsible, at
least in part, for their ability to cause neoplasia. After treatment
of mice with 3-methylcholanthrene (3-MC), dibenz~ah~anthracene, or BaP,
a prolonged depression of the immune response to sheep red cells was
noted; the noncarcinogens BeP and anthracene were ineffective in this
regard.l07'l63 The effects of the PAHs have been reviewed by
Baldwin,4 who reported a good correlation between degree of immuno-
suppression and carcinogenicity.
In
Although the previously cited work implied a link between the two
activities, Dale and Hedges37 and Stutmanl64 definitively
dissociated immunosuppression from carcinogenicity. Using guinea pigs,
Dale and Hedges concluded that the effects of the PAHs were due to
generalized toxicity and were not likely to persist long enough to lead
to neoplasia. Stutman produced tumors in mice with very low doses of
3-MC--doses that did not influence the immune status of the animals.
To conclude, some PAHs at high doses can alter the immune status of
animals when administered to the point of general toxicity, whereas
exhaust and emission have not been shown to do so.
SKIN
The major changes occurring in skin after application of emission
or PAHs are associated with neoplasia and are discussed later in this
chapter.
KIDNEY
The toxicity of diesel fuel to kidney and other tissues has been
described in only one report: a sailor cleaned his hair with diesel
fuel and was later hospitalized for renal failure.6 This acute
intoxication also resulted in damage to the liver, the gastrointestinal
tract, and the lungs. The information presented does not allow further
definition of the toxic components responsible for the pathologic
condition.
GLANDS, REPRODUCTION, AND TERATOLOGY
Although individual PAHs have pathologic effects on some glandular
tissue, little toxicity has been reported after administration of
various kinds of emission. The oral administration of 7,12-DMBA to
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female mice caused the destruction of small oocytes and reduction in
the number of growing and large oocytes.93 This compound also caused
specific destruction of the adrenal cortex in the rat.13 3-MC
administration resulted in destruction of the primordial oocytes in the
mouse.l09 3-MC or BaP given intraperitoneally produced abnormally
shaped sperm indicative of damage to the primary spe``Ilatocytes and
spermatogonia.l83
With regard to reproduction and teratology, only few PAHs have been
tested. The feeding of BaP to female rats resulted in no abnormalities
in their ovarian cycle, ovulation, fertilization, or implantation, and
few resorptions were observed in treated pregnant rats. 83 Similar
findings have been reported for the mouse.139
SHORT-TERM MODEL SYSTEMS FOR DETECTING EFFECTS
Whole-animal experiments for assessing toxicity are often expensive
and time-consuming. Therefore, alternative approaches have been
developed. A variety of short-term biologic model systems are avail-
able for assessing the effects of exhaust, its particulate components,
and pyrene analogues. These systems are characterized by the use of
multiple end points to measure genotoxicity, the use of both bacterial
and mammalian cell lines, the use of end points that can be evaluated
in relatively short periods (i.e., 1 d to 6 wk)2 and the incorporation
of an exogenous source of metabolic activation for generating the
active PAN metabolites. Each end point in concert with a particular
cell system has its own unique strengths and weaknesses. Recognizing
this fact, the regulatory agencies have required a battery of
short-term tests, to provide a more complete picture of the potential
biologic activity of a test chemical. The categories of available
short-term tests are presented in Table 4-1, with a partial list of
some of the particular tests given in Table 4-2 (see Hollstein et
_.74 for details)
Examples of the use of these tests in a short-term battery are
presented in Tables 4-3 and 4-4. Table 4-3 demonstrates the guidelines
that the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)
recommended; tests recommended by the Organisation for Economic
Cooperation and Development (OECD) are presented in Table 4-4. In
general, these batteries include the evaluation of three or more end
points from the following list: toxic effects, mutagenesis, DNA damage
and repair, chromosomal alteration, and neoplastic transformation.
TOXIC EFFECTS
Toxicity is usually manifested by such end points as cell death,
increase in generation time, decrease in respiration, decrease in rate
of macromolecular synthesis, and release of particular cell-bound
proteins. Many of these end points have been used in bacteria, 102
pro tozoa,75 algae,172 invertebrates,172 fish 148 and mammalian
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cells.88 Cell death is also used concomitantly with virtually every
assay system, to determine the numbers of cells at risk.
MIJTAGENE S I S
A mutation is any heritable change in the phenotype of an organism
or cell resulting from a change in its DNA. A mutation need not be
reflected in a change in function. The phenotypic expression of such a
change can be detected in a variety of cellular proteins. Examples of
genetic markers that use mutagenesis as the end point are given in
Table 4-5.
The bacterial systems are best exemplified by the Salmonella
typhimurium test strains developed by Ames et al.3 This system
measures the reversion rate to histidine prototrophy in five test
strains that carry specific frameshift and base-pair substitutions at
the his locus and a series of mutations at the other loci to make the
bacteria more sensitive to chemically induced mutation. The deep rough
mutation (rfa ), ultraviolet-light sensitivity (uvr B ), nitrate
reducLase deficiency (chl-), biotin deficiency (bio~), and
introduction of R factor plasmids are examples of alterations of these
test strains to make them more sensitive to chemically induced
mutagenesis. Because reversion to his prototrophy is being measured, a
battery of strains (three to five) must be tested, to ensure detection
of point mutation, frameshift mutation, and intragenic deletion. To
circumvent this problem, there have been attempts to standardize a
forward-mutation assay with S. t`~himurium. Forward mutations at both
the arabinose-resistance (grail ~ and 8-azaguanine-resistance
(8-Azr~l56 genes have been described. These assays have the
advantages of detecting virtually all mutagenic events, detecting
mutagenesis at more than one genetic locus (probably at least three),
and requiring the use of only one test strain.
Mutagenesis testing in mammalian cells has used cell types that
range from the rapidly growing, easily handled cell lines, such as
CHot55 and V-79, 20~27 to the more difficult testing of in
vivo-derived human lymphocytes .3 Advantages of the CHO and V-79
cells include high plating and cloning efficiencies, pseudodiploidy,
and the ability to monitor mutagenesis at a variety of genetic loci.
disadvantage is that these cells have little or no capacity to
metabolize xenobiotics, especially pyrenes. Recent results54 suggest
that hamster-derived cell lines, such as CHO, have limited capacity to
remove 06-alkylated guanine; thus, they may be def icient in DNA repair.
The merits and limitations of the three most widely used loci for
testing with mammlian cells are presented in Table 4-6. Such end
points as resistance to purine analogues, to 5-bromodeoxyuridine
(5-BUdR), and to ouabain collectively can detect most of the potential
genotoxic effects of PAHs.77 These end points are now being used
simultaneously to limit the possibility of false-negative conclusions.
In assays for purine-analogue resistance, mutants lacking the enzyme of
the purine salvage pathway, bypoxanthine-guanine phosphoribosyl
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transferase (HGPRT), are identified by their resistance to toxic
analogues, such as 8-azaguanine or 6-thioguanine. In assays for
ouabain resistance, mutants are detected by their ability to grow in
the presence of the glycoside ouabain. The basis of the latter muta-
tion is an alteration in the receptor for the membranal sodium-
potassium ATPase system. In the assay for 5-BUdR resistance, an
alteration of the enzyme thymidine kinase is responsible for the mutant
phenotype. The altered enzyme is unable to "activate" 5-BUdR by
catalyzing its conversion to a deoxyribonucleotide; the latter is
required for cell death.
Such cells as C3HlOTl/2 and BALB/3T3 have also been used in
mutagenesis studies; these cells have easily detectable hydrocarbon-
metabolizing activity.36~92 However, they are hypotetraploid, may
not detect some recessive mutations, and may not detect some mutations
that are expressed codominantly.95 These cells express a high degree
of contact inhibition and low saturation density and thus can be used
in bioassays of neoplastic transformation. Recent studies have
suggested that such cells can be used to detect simultaneously the
mutagenic and transforming capacities of test chemicals.35 Primary
cell strains and in vivo-derived cells have been used in mutagenesis
assays; although they have high PAH-metabolizing capacities and are
diploid, the difficulty in growing, handling, and evaluating data from
these mixtures of cells is an important disadvantage.120
DNA DAMAGE AND REPAIR
Assays for DNA damage and repair have also used both bacterial and
mammalian cells. Primary DNA damage in mammalian cells has been
measured by such end points as selective toxicity in strains of cells
deficient in ONA repair,144 increase in rate of DNA elusion under
alkaline conditions,165 formation of specific pyrene-DNA
adducts, 133 increase in rate of unscheduled DNA synthesis, 162
increase in incorporation of specific d~es,ll and increase in
incidence of sister chromatic exchange. 80 DNA repair is a specific
response to DNA damage. The covalent interaction of chemicals with ONA
provokes an enzymatic repair of the damaged regions of DNA.142
Repair synthesis can be measured in a variety of ways, but
incorporation of radioactive precursors into DNA is the
simplest.29~60 A DNA damage-repair system that shows promise in
detecting chemically induced DNA alteration uses the rat
hepatocyte.l79 This assay has the advantages of using nondividing
cells (normal semiconservative DNA replication is suppressed) and using
freshly cultured cells that have high endogenous capacity for
carcinogen metabolism or activation. It has recently been shown to be
effective in detecting the ability of a variety of chemical carcinogens
(including many different PAHs) to damage ONA.136
An increase in sister chromatic exchange (SCE) may be one of the
best measures of DNA damage in humans. This end point, which involves
incorporation of 5-BUdR into DNA during two cycles of replication and
making the two chromatics stain differently, so that exchanges of
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material are scorable, seems to develop as the consequence of
presumably long-lived DNA lesions in the S phase of the cell cycle.l26
The exact mechanism of SCE formation is not understood, although it
is well known that the frequency of SCE is increased by exposure of
cells to known mutagens in viva or in vitro.2, 127~161 In fact, a
linear correlation between mutations induced at specific loci and SCE
has been demons bated in CHO cel 1~ .20 This assay has been used to
monitor the exposure of humans to potentially harmful
chemiCa1~51, 94~ 118 and even to cigarette 9moke.76
The in viva techniques for detection of SCE can be applied in two
basic ways. One method involves 5-BUdR incorporation into bone-marrow
cell DNA by inoculation of solutions or implantations of 5-BUdR pellets
directly into animals2 and exposure of the animals to the chemical
under study; this method has been used to detect in viva DNA damage
via such substances as cyclophos~hamide,9 styrene,31 benzene,ll7
urethane,24 and cigarette smoke. The second method involves the
incorporation of 5-BUdR into lymphocyte cultures during mitogen-induced
activation in vitro; this has been used in the human studies mentioned
above. Good baseline data on the incidence and variation of SCE in
humans now exist.l8 SCE has also been shown to persist for several
days or even months after~chemical exposure and thus can serve as an
index of acute or chronic exposure to chemicals.94~118~161
Comparison of rates of formation of SCE and specific ON.A adducts
suggests that, for several types of mutagens, induction of SCE does not
necessarily result from a single specific ONA lesion.166
CHROMOSOMAL ABERRATION
Assays for chromosomal aberration are also used to monitor for the
mutagenic activity of test chemicals. These assays detect major
rearrangements in the chromosomal or chromatic structure and include
such end points as chromosomal or chromatic breaks, chromatic trans-
location, dicentric chromosomes, ring chromosomes, balanced transloca-
tion, and inversion.46~108 Another test for acutely altered chromo-
somes is the micronucleus test, in which chromosomal damage leads to
fragmentation of chromosomes or malfunction of the spindle apparatus,
so that whole chromosomes lag behind the rest and, accordingly, form
micronuclei.8l These techniques can be used with tissues derived
either in vitro or in viva much like those used for analysis of SCE.
Generally, agents that induce point mutation also induce chromosomal
aberration. In humans, mitogen-activated lymphocytes can be used to
monitor for the effects of exposure to physical and chemical agents.
Exposure to radiation, to such chemicals as alcohol and vinyl chloride,
and to cigarette smoke causes increases in chromosomal aberra-
tion.l20 Cytogenic end points of aberration are useful, but one
should remember that often chemicals induce very few aberrations at
concentrations that permit the end point of gene mutation to be readily
observed.141 In recent comparisons of three cytogenetic tests-
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induction of chromosomal aberration, induction of micronuclei, and
induction of SCE--the third proved to be the most sensitive in testing
with several PAHs.8
NEOPLASTIC TRANSFORMATION
-
Neoplastic transformation has been assayed by a variety of in vitro
systems, and it is not possible to review all the pertinent literature
here. The reader is directed to the recent reviews of Casto and
Carver,21 Heidelberger,64 and Mishra et al.115
Specific cells that have been used for assay of in vitro chemically
induced neoclassic transformation include normal rodent (di laid) cell
strains,42, 29 established aneuploid rodent cell lines,36~43~64~84
cell lines derived from human tumors,80~138 and cell lines initiated
from apparently healthy human tissue.50~85~113~154 Table 4-7 com-
pares properties of some mammalian-transformation systems. These cell
lines share the following properties to some degree: They exhibit
density-dependent inhibition of cell division and reach a defined
saturation density, do not form colonies on soft agar or agarose, and
do not give rise to tumors when inoculated into immunosuppressed
syngeneic hosts. After transformation by chemicals, they lose the
density-dependent inhibition of cell division and form piled-up,
cries-crossed foci; they grow on soft agar or agarose, and they form
tumors when inoculated into host animals. In addition many trans-
formed cells exhibit increased fibrinolytic activity, 134 altered
morphology in the scanning electron microscope, 106 specific chromo-
somal arrangement, 10~134 and specific DNA sequences that can be
transfected into normal cells, resulting in formation of the trans-
formed phenotype. 33, 152 Although each of these cell systems has been
successfully used to ascertain the biologic activity of chemical
agents, none appears to be capable of universally detecting all classes
of chemical carcinogens, low concentrations of all such agents, and
relatively weak biologic activity of some chemicals.
MUTAGENESIS
-
As just discussed, a number of model systems are available for
assessing the mutagenic activity of emission, individual PAHs, and
their mixtures. These are in two categories: bacterial systems and
mammalian cell-culture systems. The activity of emission and its PAR
constituents is discussed below relative to both kinds of model.
BACTERIAL MUTAGENESIS
Particulate matter from city air has been tested for mutagenic
activity with the Salmonella/microsome system.131~167~169 In all
cases, a positive response was obtained. Furthermore, many of the
samples exhibited direct-acting mutagenic activity, i.e., the addition
of activating enzymes present in a liver S-9 fraction was not required
4-10
Representative terms from entire chapter:
polycyclic aromatic
