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OCR for page 555
Potential Carcinogenic
Effects of Polynuclear
. ·
Aromatic psycho car cons
anc" N~troaromatics in
Mobile Source Emissions
STEPHEN S. HECHT
American Health Foundation
Historical Perspective and Directions for the Future / 556
Evaluation of Polyaromatic Hydrocarbon and
Nitro-Polyaromatic Hydrocarbon Carcinogenicity / 556
Role of Polyaromatic Hydrocarbons as Human Carcinogens / 556
Tumorigenicity of Polyaromatic Hydrocarbons in Laboratory
Animals / 558 Modifiers of Polyaromatic Hydrocarbon
Carcinogenesis / 561 Carcinogenicity of Nitro-Polyaromatic
Hydrocarbons / 562
Metabolic Activation and Detoxification of Polyaromatic
Hydrocarbons and Nitro-Polyaromatic Hydrocarbons / 563
Absorption and Distribution upon Inhalation / 563
Benzotaipyrene / 564 Other Polyaromatic Hydrocarbons / 566
Nitro-Polyaromatic Hydrocarbons / 567
Research Problems Relating to the Potential Carcinogenic Effects
of Polyaromatic Hydrocarbons and Nitro-Polyaromatic
Hydrocarbons in Humans / 568
Individual Dosimetry / 568 Bioassays in Laboratory
Animals / 570 Mechanisms of Polyaromatic Hydrocarbon and
Nitro-Polyaromatic Hydrocarbon Carcinogenesis / 571
Summary / 572
Summary of Research Recommendations / 573
Air Pollution, the Automobile, and Public Health. (~3 1988 by the Health Effects
Institute. National Academy Press, Washington, D.C.
555
OCR for page 556
556
Effects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
Historical Perspective and
Directions for the Future
Pott first observed an association between
soot and cancer in 1775 (Pott 1775), and by
the early twentieth century it was clear that
soot, coal tar, and pitch could cause cancer
in humans (International Agency for Re-
search on Cancer 1985a). In the 1930s,
pioneering studies by Kennaway and
Hieger (1930) and Cook et al. (1933)
established that polynuclear aromatic hy-
drocarbons (PAHs) were carcinogenic com-
ponents of pitch. Although the following
50 years have brought major advances in
our understanding of the mechanisms by
which PAHs can cause cancer, our ability
to assess the health effects-and in particu-
lar the potential carcinogenic effects of
PAHs in humans, remains incomplete. The
aspects of PAH carcinogenesis summarized
in this chapter include epidemiologic stud-
ies that may link PAHs to human cancer,
. . . . . .
carc1nogenlc1ty assays 1n .a ooratory an1-
mals, key features of PAH metabolic acti-
vation and detoxification, and the effects of
modifiers on these processes.
In contrast to the PAHs, known for half
a century, nitro-substituted PAHs (nitro-
PAHs) have only recently been recognized
as environmental carcinogens, whose pres-
ence in diesel exhaust is of particular con-
cern (Schuetzle 1983~. Although less is
known about their health effects than about
those of PAHs, it is clear that some nitro-
PAHs are potent mutagens and carcino-
gens.
Using these studies as a base, we identify
significant gaps that detract from our abil-
ity to assess PAH Carcinogenicity in hu-
mans, summarize data on the carcinogenic
effects and metabolism of nitro-PAHs in
laboratory animals, and suggest directions
for future research.
Recent rapid progress in research on
PAHs and nitro-PAHs is due to improved
analytical and spectroscopic techniques as
well as to major advances in molecular
biology. Those new techniques, which
may soon permit the measurement of an
effective biological dose of a carcinogen for
humans, combined with animal experi-
ments, may make it possible to develop
indicators of individual susceptibility to
PAH or nitro-PAH carcinogenesis. These
exciting developments represent an impor-
tant frontier in chemical carcinogenesis re-
search, and their application to assessing
the health risks of PAHs and nitro-PAHs
are a focus of this chapter.
Evaluation of Polyaromatic
Hydrocarbon and Nitro
Polyaromatic Hydrocarbon
Carcinogenicity
Role of Polyaromatic Hydrocarbons
as Human Carcinogens
Since the PAHs to which humans are ex-
posed always occur in a mixture of many
compounds, some potentially carcinogen-
ic, assessing PAHs as human carcinogens
by epidemiologic studies is difficult. The
International Agency for Research on Can-
cer (IARC) has evaluated Carcinogenicity of
such mixtures and published results in the
IARC Monographs on the Evaluation of the
Carcinogenic Risk of Chemicals to Humans.
. . . . . .
~V1C .ence or carclnogen1c1ty rom stuc 1es
in humans is categorized as follows: (1)
"sufficient evidence" indicates a causal re-
lation between the agent and human cancer;
(2) "limited evidence" indicates a credible
causal relation without excluding other ex-
planations; (3) "inadequate evidence" indi-
cates either that pertinent data are few or
that the available data do not exclude a
presumed chance association; and (4) "no
evidence" indicates that adequate studies
are available and these show no evidence of
Carcinogenicity Other working groups
have also considered the possible roles of
PAH-containing mixtures in human cancer
etiology. Studies of sources, other than
automotive, implicate PAHs as human car-
cinogens and are discussed below. Evidence
for the role of mobile sources is discussed in
Carcinogenicity of Nitro-PAHs.
Tobacco Smoke. Sufficient evidence now
indicates that tobacco smoke is carcino-
genic to humans, that malignant tumors of
the respiratory tract and upper digestive
system are causally related to smoking to-
bacco, and that malignant tumors of the
bladder, pancreas, and renal pelvis specifi
OCR for page 557
Stephen S. Hecht
557
cally are causally related to cigarette smok-
ing (International Agency for Research on
Cancer 1986~. The 1982 report by the Sur-
geon General of the United States con-
cluded that "cigarette smoking is the major
single cause of cancer mortality in the
United States. Tobacco's contribution to
all cancer deaths is estimated to be 30
percent" (U.S. Department of Health and
Human Services 1982a). Evidence increas-
ingly suggests that passive exposure to
tobacco smoke, as in polluted indoor envi-
ronments, may increase the risk of lung
cancer (International Agency for Research
on Cancer 1986~.
P`AHs are important carcinogenic con-
stituents of the particulate phase of cigarette
smoke in concentrations ranging from 1
to 60 ng/cigarette (U. S. Department of
Health and Human Services 1982b), and
their tumorigenic activities are enhanced by
other agents in tobacco smoke. It is very
likely that they are involved in the cancer-
causing properties of tobacco smoke, but
their role is difficult to assess because of the
many other toxic and carcinogenic constit-
uents of tobacco smoke, such as nitrosa-
mines whose concentrations exceed those
of PAHs (Hoffmann and Hecht 1985), vol-
atile aldehydes, and aromatic amines.
.
Coal Tars, Shale Oils, and Soots. An
IARC Working Group (1985a) concluded
that occupational exposure to coal tars is
causally associated with incidence of skin
cancer in humans, and that coal tar pitches
are carcinogenic in humans. They noted
that a cohort study of U.S. roofers indi-
cated a greater risk of lung cancer and other
cancers. Among the 10,000 compounds
that may be present in coal tars, PAHs
occur in concentrations ranging from 0.1 to
10 percent in high-temperature coal tars,
and certainly contribute largely to the ob-
served carcinogenic properties of coal tars.
Other IARC groups (1985b,c) have con-
cluded that there is sufficient evidence that
shale oils and soot are carcinogenic to hu-
mans and contain relatively high levels of
PAHs.
Coal Gasification and Coke Production.
The IARC (1984c) has concluded that cer-
tain exposures in the retort houses of older
coal gasification processes and in the coke
production industry are carcinogenic in hu-
mans. The relative risk of lung cancer is as
high as 16-fold in topside coke oven work-
ers with 15 years or more of exposure.
Consideration of duration and location of
employment in the plant has shown a dose/
response relationship for lung cancer. In an
association between mortality from lung
cancer and exposure to coal tar pitch vola-
tiles, PAHs are again likely prominent
causative agents (Redmond 1983~.
Aluminum Reduction and Iron and Steel
Founding. Although an IARC Working
Group (1984b) found only limited evidence
of increased incidence of cancer in alu-
minum production and iron and steel
foundry workers, some evidence links alu-
minum production to bladder cancer. Lev-
els of total PAHs in aluminum production
range from <1 to 2,800 ,ug/m3, determined
by personal sampling for 2~ hr at various
sites. PAHs or their metabolites have also
been detected in the urine of exposed work-
ers (Becher and Bjorseth 1983; Interna-
tional Agency for Research on Cancer
1984b).
Mineral Oils. Mineral oils used in such
occupations as mulespinning, metal ma-
chining, and jute processing have been
found to be carcinogenic to humans, and
exposures have consistently been linked to
cancer of the skin, and particularly of the
scrotum. The levels of PAHs present in
such oils vary, depending on source and
processing (International Agency for Re-
search on Cancer 1984a).
Urban Pollution. In contrast to certain
occupational exposures, epidemiological
evidence indicates that after correction for
smoking and occupation, exposure to gen-
eral air pollution (defined as a body of
contaminated air extending over a popula-
tion area of appreciable size) has little, if
any, effect on rate of death from lung
cancer (Hammond and Garfinkel 1980~.
Conclusion. Although none of the studies
mentioned here has specifically incrimi-
nated PAHs as causative agents of cancer,
the overall data, together with the results
OCR for page 558
558
Anthanthrene
2
904 8~2
7 6 6 S 4
Benzo[a]fluorene Benzolbifluorene
~ 2 3
2 1O 1 12 Q
11 - 6 7~3 lO - S
9 8 7 6 S 8
Effects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
7~2 10~?4
5 10 4 8 7 6
Anthracene
Benzo[b]fluoranthene Benzo[ghi]Duoranthene Benzoij]fluoranthene
10~4
8 7 6
Benzo[k]fluoranthene Benzolghi]perylene
12 ~
~2
7 6
Chrysene
2 12 ~
[~5 ^3
7 6 7 6 S
fluoranthene
Indenol1,2,3-cd]pyrene
10 ~10 1
8~3 8~3
6 6 5
Phenanthrene Pyrene
tested most extensively on mouse skin,
since such assays are convenient and inex
pensive. Some compounds have been
tested by other protocols, including subcu
Benzla]anthracene
taneous Injection in mice and rats, 1ntrapul
1 2 3 monary injection in rats, and intratracheal
10~4 instillation in hamsters. Tables 1 and 2
IS summarize an IARC Working Group eval
senzOlc]f~uOrene nation of carcinogenicity of these PAHs in
laboratory animals.
10 ~
8~3
6 S 4
Cyclopentalcd]pyrene
Perylene
Figure 1. Structures of some PAHs commonly de-
tected in exhaust emissions.
of bioassays described below, strongly in-
dicate that PAHs can cause cancer in hu-
mans.
Tu me rigenicity of Polyaro ma tic
Hydrocarbons in Laboratory Animals
Figure 1 illustrates the structures of the
PAHs commonly detected in exhaust emis-
sions (International Agency for Research
on Cancer 1983~. Methylchrysenes as well
as methyl- and dimethylphenanthrenes are
also detectable. To evaluate carcinogenic
potential, these compounds have been
Mouse Skin Bioassays. Two protocols
are used. The first, the initiation/promo
tion protocol, consists of the application of
a single large dose or series of smaller doses
of the PAH to the skin, followed by re
peated application of the tumor promoter
12-O-tetradecanoylphorbol-l~acetate (TPA).
(A tumor promoter is a substance that
Benzo~a~pyrene does not itself induce tumors, but when
applied after a tumor initiator, for ex
ample, a PAH, enhances its activity.) The
second, or complete carcinogenicity pro
tocol, consists of repeated applications of
the PAH to the skin. Frequently, the results
of both protocols agree (LaVoie et al.
1979~.
Among the unsubstituted PAHs, signif
icant activity is observed only in com
pounds with four or five aromatic rings.
Benzo~aipyrene (BaP) and benzo~b]fluo
2 3 ranthene are the most tumorigenic, fol
~ ~ r~4 lowed by benzo~fluoranthene. Weak
,O~s tumor~gen~c~ty has been observed for benz
8~6 [ajanthracene, benzo~k]fluoranthene, chrys
Tripheny~ene ene, cyclopenta~cdipyrene, and indeno
[1,2,3-cd~pyrene. Methyl substitution can
significantly alter tumorigenicity. As
shown in table 2, 5-methylchrysene is a
potent tumorigen, with activity similar to
that of BaP, and 1,4- and 4,10-dimethyl
phenanthrene also are relatively strong
tumor initiators. For a detailed review
of structure/activity relationships among
methylated PAHs, see Hecht et al. (1988~.
Respiratory Tract Bioassays. These pro
tocols are relevant to the problem of human
respiratory exposure to PAHs, and include
intratracheal instillation, lung implanta
tion, and inhalation. Intratracheal instilla
tion of PAHs has been used most exten
sively with the Syrian golden hamster
OCR for page 559
Stephen S. Hecht
559
Table 1. Representative Tumor-Initiating Activity of PAHs on Mouse Skill and IARC
Evaluations of Carcinogenicity of Parent PAHs Commonly lDctectcd in Exhaust Emissions
Representative Assays of Tumor-lnitiati~;,
Activity on CD-I Mouse Skin
Compound
Dose
(nmole)
TEA
(%)
7
14
23
10
9()
25
60
T/A
Pcfcrcncc
IA1lC Evaluation
of Carcinogel1icity
in Laboratory
Animals''
Anthanthrene
Anthracene
Benz[a janthracene
Benzota]fluorene
Benzotb]fluorene
Benzote]fluorene
Benzotb]fluoranthene
Benzotyhi]fluoranthene
Benzot]fluoranthene
Benzo~k]fluoranthene
Benzotghi~perylene
Benzo~a~pyrene
Benzote~pyrene
Chrysene
Coronene
Cyclopentatcdipyrene
Fluoranthene
Indenot1,2,3-edipyrene
Perylene
Phenanthrene
Pyrene
Triphenylene
91()
10,0()()
2,00()
4,63()
4,63()
4,63()
119
NT
119
119
910
119
6,()0()
4,390
1,670
2,5()0
4,950
900
3,970
5,620
4,950
NT
().1 HofEmallll and Wynder (1 t)66)
().1 Scribl,cr (1973)
0.3 Wood ct al. (1977)
(). ~LaVoi˘: ct al. (1 t)X 1 a)
().4 LaVoic ct al. (19X1 a)
0.3 LaVoic ct al. (1981 a)
0.3 LaVoie ct al. (19X9b)
3()
85
14
55
30
37
3
17
5
()
().6
().1
0.1
4.9
O.1
().6
().5
o
().3
().1
()
O.1
LaVoic ct al. (19X2b)
LaVoic ct al. (19X9b)
Hoffman and Wyndcr (1966)
LaVoie et al. (19X9b)
Bucning et al. (19X())
Hccht et al. (1974)
Van Dunrcn ct al. (196X)
Wood et al. (198())
HofEmanll ct al. (1 t)79)
HofFmann and Wynder (1966)
El-Bayoumy ct al. (19X9)
LaVoic~ et al. (19X1 b)
El-Bayoumy ct al. (19X'))
Limited
None
Sufficient
adequatc
Illadequatc
Inadcq~atc
Sufficient
Inadequate
Suff~cicl~t
Suff~cicnt
ll~adeq~atc
Suff~cicllt
Inadeq~atc
Limited
Inadcq~ate
Limited
N Ol1C'
Sufficient
Inadcquatc
Inadcq~atc
North
Inadequate
~ From International Agency for Research on Cancer (1983).
b It was not active as a complete carcinogen On mouse skin (Wyndcr and Hoffman 1959).
' A study has shown that fluoranthenc is tumorigenic in newborn mice (Busby et al. 1~)84).
NOTE: NT = not tested as a tumor initiator; T/A = tumors per animal; TEA = tumor-beari~,g animals.
because it has no spontaneous lung tumors
and is resistant to pulmonary infection and
inflammation. Tumors are induced by in-
stillation of the PAH and a carrier, for
example, ferric oxide, (Fe2O3) or in a sus-
pension in saline (Stinson and Saff~otti
1983~. Squamous cell carcinoma of the
trachea and bronchi induced in a high per-
centage of Syrian golden hamsters given
intratracheal instillations of BaP closely re-
semble human tumors.
Although the carcinogenic effects of BaP
in the hamster respiratory tract have been
studied, investigations of other PAHs have
been few. Sellakumar and Shubik (1974)
found that when either benzotb]fluoran-
thene, dibenz~a,hianthracene, benz~ajan-
thracene, or pyrene was instilled with
Fe2O3 in the hamster trachea, the incidence
of respiratory tumors was insignificant.
However, in this model system diben-
zota,i~pyrene and dibenzo~c,gicarbazole
were highly tumorigenic. In experiments
with other species, Hirao and coworkers
(1980) found that BaP in saline caused lung
cancer in rabbits, and Yoshimoto and co-
workers (1977) observed that BaP with a
carrier induced tumors in mice and rats.
Stanton and coworkers (1972) implanted
PAHs dissolved in a mixture of beeswax
and trioctanoin in rat lungs and observed
the development of epidermoid lung carci-
noma. Deutsch-Wenzel and coworkers
(1983) tested a variety of PAHs using this
protocol, and found that BaP was the most
tumorigenic PAH of those commonly
detected in exhaust emissions, while an-
thanthrene, benzo~b]fluoranthene, and in-
deno~l,2,3-cdipyrene were moderately tu-
morigenic. However, the tumorigenicity
OCR for page 560
560
Effects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
Table 2. Representative Tumor-Initiating Activity on Mouse Skin and IARC Evaluations of
Carcinogenicity of Methylated Chrysencs and Phc~anthrc~cs
Rcpresentativc Assays of Tun~or-Initiating
Activity on Mouse Skill
Dose
Parent System Isomer (nmole)
Methyl
TEA (%)
T/A
IAllC Evaluations of
Carc~,ogen~c~ty ~,
Laboratory Animals"
Chryseneb
Phenanthrene'
2
3
4
6
2
3
4
9
1,4
1,9
2,7
3,6
4,5
4,9
4,10
4, 1 3()
4,13()
4,13()
1, 94()
41()
4, 1 3()
4,13()
1, 94()
41()
1()()
33
4,130
5,910
5,91()
5,910
5,910
5,91()
4,85()
1 ,460
4'85()
4,85()
4,85()
4,850
4,85()
4,850
1,460
3()
49
7()
2()
15
35
~5
10()
1 O()
9()
8()
35
()
()
i)
()
()
10()
8()
()
5
()
5
1()
55
35
.3
().7
1.3
().4
().5
4.S
S.(
5.5
c ~
J. _
3.9
().6
()
()
()
()
()
5.3
3.9
()
().1
()
().1
().1
1.5
1.6
Illadeqelatc
Limitcd
Limitcd
Limitcd
SufElcicl~t
Linlited
Inadcquatc
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
~ From International Agency for Research Ol1 Cancer (19~33).
b Hecht et al. (1974).
c LaVoie et al. (1981b, 1982a).
NOTE: NC = not considered; T/A = tumors per animal; TBA = tumor-bearing animals.
Of anthanthrene was higher than expected squamous cell carcinoma of the lung
on the basis of mouse skin studies. (Laskin et al. 1970~. In another experiment,
Kendrick and coworkers (1974) found A' ' ' '~' '
that instillation of PAHs into subcutaneous
tracheal transplants in rats results in hyper
plasia, dysplasia, squamous metaplasia, and
squamous cell carcinoma. Benzotaipyrene,
but not benzo~eipyrene (BeP), was carcino
genic in that system. However, Topping
and coworkers (1981) found that BeP was
cocarcinogenic for the connective tissue,
but not the tracheal epithelium.
Because of their expense, inhalation ex-
periments with PAHs have been limited. In
one experiment, 2 of 21 rats housed in fresh
air and 5 of 21 rats housed in an atmosphere
containing sulfur dioxide (SO2) developed
1 hyssen and coworkers (~) observed
exposure-related neoplasms in the nasal
cavity, larynx, pharynx, esophagus, and
forestomach of Syrian golden hamsters ex-
posed to BaP.
Dose/Response Relationships. Clear dose/
response relationships for BaP-induced tu-
mor formation have been demonstrated by
using the Syrian golden hamster intratra-
cheal instillation model and the mouse skin
initiation/promotion protocol. The yield of
respiratory tract tumors, tumor latency,
and tumor multiplicity related to dose in
hamsters treated with BaP and Fe2O3 (Saf
OCR for page 561
Stephen S. Hecht
561
fiotti et al. 1972a,b), or BaP in buffer and
physiological saline (Ketkar et al. 1979),
depended on the number of administra-
tions, the dosage per administration, and
the fractionation of doses.
Similar effects have been observed on
mouse skin (Saffiotti and Shubik 1956~. In
the mouse skin initiation/promotion proto-
col, a linear dose/response relationship for
papilloma formation in Sencar mice ranged
between 100 and 600 nmole/mouse; at
higher doses the tumor response leveled off
(Ashurst et al. 1983~. The effects of differing
initiating doses of 7,12-dimethylbenz~a]-
anthracene showed that linear extrapolation
from high doses may lead to underesti-
mation of low-dose tumor risks (Stenback
et al. 1981~.
Effects of Mouse Strain. Aryl hydrocar-
bon (Ah)-responsive mice (for example,
C57BL/6N) are more susceptible to tumor
induction from subcutaneous injection of
BaP, 3-methylcholanthrene, or dibenz-
[a,hianthracene than are Ah-nonresponsive
mice (for example, DBA/2N). These dif-
ferences have been attributed to the dif-
fering abilities of these mice to metabolize
the PAHs to their ultimate carcinogenic
forms. Inducibility of the cytochrome
P-450 enzymes that metabolize PAHs ap-
pears to be related to PAH tumorigenicity
(Nebert 1981~. The Ah gene codes for a
cytosolic receptor which can bind the in-
ducer, such as 3-methylcholanthrene. The
inducer/receptor complex is translocated
into the nucleus and in some way initiates
increased P-450 synthesis.
Modifiers of Polyaromatic
Hydrocarbon Carcinogenesis
The influence of modifiers is perhaps one of
the most important but least well under-
stood areas of cancer induction by PAHs.
Modifiers can be broadly classified as either
promoters, cocarcinogens, or inhibitors of
carcinogenesis. Promoters are generally
noncarcinogenic substances which, when
applied subsequently to PAHs, will cause
tumors. In the case of the most widely
studied promoter, TPA, tumors can be
induced on mouse skin even if the TPA is
applied one year after administration of
7,12-dimethylbenz~ajanthracene (Van Du-
uren et al. 1975~.
This observation supports the concept
that initiation, even by a single dose of a
PAH, is essentially irreversible, consistent
with a change in DNA. Exposure to a
single dose of a PAH can initiate cells but
may not cause tumors, but exposure of
initiated cells to multiple doses of a pro-
moter can lead to tumor development.
Thus, promotion may be important in de-
termining whether or not environmental
exposure to PAHs results in tumor devel-
opment. TPA is an exceptionally effective
tumor promoter for experimental studies,
but it does not occur in significant quanti
.
ties in the environment.
The most important known environ-
mental tumor promoters are tobacco
smoke and diet. The tumor-promoting ac-
tivity of tobacco smoke and its condensate
has been clearly demonstrated in studies
using PAHs as initiators in either the mouse
skin or the intratracheal instillation models
(Hoffmann et al. 1978~.
.. . .. .
Epidemiologic
stud~es ~nd~cate that cessation of cigarette
smoking leads to a lower risk for lung
cancer, consistent with reversibility of pro-
motion (Wynder and Hoffmann 1979~. Ex-
tensive studies with the 7,12-dimethylbenz-
[ajanthracene-induced Sprague-Dawley rat
breast tumor model have demonstrated
that a high-fat diet can promote breast
tumor development. This is also in agree-
ment with some epidemiologic studies
(National Research Council, Committee
on Diet, Nutrition, and Cancer 1982~.
Cocarcinogens are defined as substances
that enhance tumorigenicity when admin-
istered simultaneously with a carcinogen.
Important environmental cocarcinogens in-
clude tobacco smoke, polyphenols, and
PAHs. The cocarcinogenicity of tobacco
smoke is due to its neutral polar and weakly
acidic fractions (Hoffmann et al. 1978~.
Investigations of the compounds responsi-
ble for this activity have led to the identi-
fication of a number of cocarcinogens that
are also environmental or dietary constitu-
ents. Important among these is catechol,
which is strongly cocarcinogenic with BaP
on mouse skin (Van Duuren and Gold
OCR for page 562
562
Effects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
Schmidt 1976; Hecht et al. 1981~. Com-
pounds related to catechol occur widely in
the diet, and conjugates of catechol are
excreted in normal human urine (Carmella
et al. 1982~.
The cocarcinogenic activities of PAHs
are particularly important because PAHs
always occur as mixtures in the environ-
ment. Pyrene, benzo~eipyrene, and flue-
ranthene are all essentially nontumorigenic,
but they all enhance the tumorigenicity of
BaP (Van Duuren and Goldschmidt 1976;
Hoffmann et al. 1978~. Other cocarcino-
gens include benzo~ghi~perylene, decane,
undecane, 4,4'-dichlorostilbene, 1-methyl-
indole, and 9-methylcarbazole (Van Duu-
ren and Goldschmidt 1976; Hoffmann et al.
1978~. Such interactions have to be consid-
ered when assessing human risk for cancer
development upon exposure to PAHs.
A broad spectrum of compounds, many of
them dietary, are capable of inhibiting PAH
tumorigenesis. These include a variety of
phenols, phenolic antioxidants such as buty-
lated hydroxyanisole (BHA) and butylated
hydroxytoluene (BHT), naturally occurring
dietary indoles and isothiocyanates, various
flavones, selenium salts, protease inhibitors,
retinoids, and carotenes (Slaga and DiGio-
vanni 1984; Wattenberg 1985~.
Inhibition has been observed in various
model systems including mouse skin,
mouse forestomach, mouse lung, and rat
breast. A number of different protocols
have been used, and the timing of admin-
istration of inhibitor versus administration
of PAH can be critical in determining
whether inhibition of tumorigenesis is ob-
served. The identification of naturally oc-
curring and synthetic inhibitors of carcino-
genesis offers promise for prevention of
PAH carcinogenesis. However, the influ-
ences of chemopreventive agents on PAH
carcinogenesis are complex. For example,
BHA is carcinogenic under certain condi-
tions, and in some experimental protocols,
BHA as well as BHT can act as tumor
promoters (Ito et al. 1985~.
Carcinogenicity of Nitro
Polyaromatic Hydrocarbons
Whereas the carcinogenic activities of
PAHs have been evaluated extensively, re
search on nitro-PAHs is relatively limited.
The observation that nitro-PAHs appear to
account for a major portion of the direct-
acting mutagenicity of diesel exhaust par-
ticulates has caused interest in their poten-
tial carcinogenic effects. No epidemiologic
studies are available on the potential carci-
nop;enicity of the nitro-PAHs that have
been identified in diesel exhaust; these in-
clude 1-nitropyrene, dinitropyrenes, hy-
droxynitropyrenes, methyl nitropyrenes,
3-nitrofluoranthene, 2-nitrofluorene, 9-ni-
troanthracene, and 6-nitrobenzo~a~pyrene
(Schuetzle 1983~.
With respect to occupational exposure to
diesel exhaust, "excess risk of cancer of the
lung, or of any other site, has not been
convincingly demonstrated" (National Re-
search Council, Health Effects Panel of the
Diesel Impacts Study Committee 1981~.
However, the Committee found fault with
the studies that have been performed and
called for additional carefully controlled
studies of populations occupationally ex-
posed to diesel engine exhaust.
Indeed, two recent studies of motor ex-
haust-related occupations and bladder can-
cer, based on the National Bladder Cancer
Study, indicated that males usually em-
ployed as truck drivers or deliverymen had
a statistically significant 50 percent increase
in risk of bladder cancer (Silverman et al.
1983, 1986~. The authors speculated that
nitro-PAHs might be involved as causative
agents. PAHs as well as nitro-PAHs are
present in motor exhaust of various types.
Experimental studies of nitro-PAH tu-
morigenicity are summarized in table 3.
1-Nitropyrene, one of the predominant ni-
tro-PAHs in diesel exhaust, induces tumors
at the site of subcutaneous application as
well as in the breast in newborn CD rats. It
also is moderately tumorigenic in the A/J
mouse lung adenoma assay. However, it
appears to be inactive in adult CD rats and
is weakly active or inactive in a number of
other experimental models.
In contrast to 1-nitropyrene, the dini-
tropyrenes are highly tumorigenic. They
have induced high incidences of subcutane-
ous tumors in rats (Ohgaki et al. 1984,
1985) and in mice (Tokiwa et al. 1985), and
1,6-dinitropyrene has caused lung carcino-
mas in 9~100 percent of Syrian golden
OCR for page 563
Stephen S. Hecht
Table 3. Nitro-PAH Tumorigenicity Assays
Compound Test System
1-Nitropyrene
2-Nitropyrene
~Nitropyrene
3-Nitrofluoranthene
6-Nitroehrysene
Newborn CD rat
A/J mouse
Mouse skin
F344/I)u Crj rat
Newborn mouse
BALB/c mouse
CD rat
CD rat
CD rat
Newborn mouse
A/J mouse
Mouse skin
Newborn mouse
6-Nitrobenzo[a~pyrene Mouse skin
Newborns mouse
Mouse skirt
Newborns mouse
F344/l)u Crj rat
Newborn mouse
F344/l)u Crj rat
Syrian golden hamster
3-Nitroperylene
7-Nitrobenz [a] anthraeene
1, 3-Dinitropyrene
1, 6-Dinitropyrene
1, 8-Dinitropyrene
2-Nitrofluorene
5-Nitroaeenaphthene
BALB/c mouse
Newborn mouse
F344/I)u Crj rat
Holtzma~, rat
Wistar rat
Syrians golden hamster
F344 rat
B6C3F, mouse
hamsters treated by intratracheal instilla-
tion (Takayama et al. 1985~. In the new-
born mouse, 6-nitrochrysene was excep-
tionally tumorigenic (Busby et al. 1984;
Wislocki et al. 1985) whereas a number of
other nitro-PAHs were weakly tumori
. . .
gen1c or inactive.
Taken together, these studies show that
nitro-PAHs are carcinogenic in laboratory
animals. Their activities depend greatly on
structure and on the model system used.
Further studies are necessary to define more
clearly the structural requirements for tum-
origenicity of nitro-PAHs as well as the most
appropriate model systems for their bioassay.
563
Result
Subcutaneous and
mammary tumors
Lung adenonlas
. . · ~ r
i .llslgulrlcallt activity
Ir
1lslgn~rlcallt activity
Liver tumors
r
Inslglllrlcallt activity
· · r
1lslgnlrlcallt activity
lnsigllificallt activity
Mammary tumors
Liver and lung tumors
Lung adeuomas
Skin tumors
Lung and liver tumors
Insignificant activity
Liver tumors (weak)
Skill tumors
Liver tumors (weak)
Subcutallcous tumors
.
Llvcr tumors
Subcutallcous fore
Lung tumors alla
Icukemia
Subcutanco~ls torpors
Liver tumors
Subcutaneous tulllors
Forcstonlach carcinoma
Ear duct, mammary,
small intcstillc tumors
Cholallgiomas
Ear duct, mammary, lung
tumors
Liver tumors
Itcfcrcnce
Hirose et al. (1~)~)
El-13ayo~lmy et al. (1')~34a)
El-Hayo~ln~y et al. (19~3~))
Nesl~ow et al. (I')X4)
Ohgaki ct al. (1~385)
Wislocki et al. (It)Xo)
Tokiwa et al. (1984)
Inlaida et al. ( I two)
In~aida et al. (19X5)
Imaida et al. (13X5)
Wislocki et al. (I')X5)
El-Bayoumy et al.
unpublished
observations
El-Bayounly et al. (19~3~))
Busby ct al. (19~34)
Wislocki et al. (19~35)
El-Bayo~ln~y et al. (I'M)
Wislocki et al. (1)X5)
El-Bayoun~y et al. (Itchy)
Wislocki et al. (I')X5)
Ohgaki ct al. (19X5)
Wislocki et al. (I'JX5)
Ohgaki ct al. (1'JX5)
Takayama et al. (13X5)
Tokiwa et al. (1'3X4)
Wislocki et al. (lC)X5)
Ohgaki Ct al. (19X4)
Miller Ct al. (1')55)
TakClll~lra Ct al. (1'374)
Takenlura et al. (1974)
National Callcer Institute
(1 t)78)
Metabolic Activation and
Detoxification of Polyaromatic
Hydrocarbons and Nitro
Polyaromatic Hydrocarbons
Absorption and Distribution
upon Inhalation
Clearance of BaP from the respiratory tract
is slower when it is associated with particles
than when it is in a pure form (Stinson and
Saff~otti 1983~. The major route of BaP
excretion is in the feces (Heidelberger and
Weiss 1951~. The results of a comparative
inhalation study by Sun and coworkers
OCR for page 564
564
Elects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
(1982) suggests that most of a BaP aerosol
was cleared by absorption into blood fol-
lowed by biliary and fecal excretion,
whereas in animals exposed to particle-
associated BaP, a substantial amount of the
lung clearance occurred by mucociliary
clearance and ingestion. These studies dem-
onstrate the importance of particles to the
in vivo fate of inhaled BaP, and they are
clearly relevant to evaluating health risks of
environmental exposure to PAHs.
Using a similar approach, Sun and co-
workers (1983) studied the fate of 1-ni-
tropyrene. In contrast to the results ob-
tained with BaP, no apparent differences in
lung retention were observed between pure
and particle-associated material. However,
the rats exposed to 1-nitropyrene coated on
particles excreted the majority of the dose
in the feces whereas those exposed to the
1-nitropyrene aerosol excreted most of the
dose in the urine. The excretion in the feces
is consistent with mucociliary clearance and
subsequent ingestion, since 1-nitropyrene
and its metabolites administered by gavage
are excreted primarily in the feces (El-
Bayoumy and Hecht 1984a).
Benzota~pyrene
It is now generally accepted that modifica-
tion of DNA is a key step in the initiation
of the carcinogenic process. PAHs, like
many other carcinogens, do not themselves
react covalently with DNA, but require
metabolic activation to reactive species.
Thus, they are considered procarcinogens.
Metabolites that are on the pathway to
reaction with DNA are called proximate
carcinogens and those that react with DNA
are termed ultimate carcinogens. The latter
are electrophiles and are formed as interme-
diates in the normal response of the orga-
nism to foreign compounds, which is gen-
erally to convert them to more polar forms
that are readily excreted (see LaVoie and
Hecht 1981~.
Major metabolic transformations of BaP
are summarized in figure 2. The metabo-
lism of BaP has been extensively investi-
gated in various systems, and reviews of its
metabolic activation and detoxification are
available (Gelboin 1980; LaVoie and Hecht
1981; Conney 1982; International Agency
for Research on Cancer 1983~. The path-
way BaP > BaP-7,8-epoxide ~ BaP-7,8-
diol > BaP-7,8-diol-9,10-epoxide is gener-
ally considered to be the major activation
pathway in BaP-induced tumorigenesis.
All other metabolic pathways illustrated in
figure 2 are generally thought to be detox-
Cation routes.
These generalizations are useful for con-
sidering the effects of BaP in various tissues
and species, but it is becoming increasingly
clear that they are oversimplifications.
There are probably aspects of BaP metab-
olism other than dial epoxide formation
that contribute to its tumorigenic activity.
DNA adducts are also formed from metab-
olites other than the dial epoxides, such as
the 4,5-epoxide of 9-hydroxy-BaP, and a
number of unidentified BaP/DNA adducts
are produced in cultured rat mammary cells
in vitro and following direct application of
BaP to rat mammary glands in vivo (Phil-
lips et al. 1985~. In addition, major uniden-
tified material that elutes rapidly from
chromatographic columns and is indicative
of unknown DNA adducts has been ob-
served in virtually all studies of BaP/DNA
adduct formation.
Factors that Influence Metabolism. The
metabolism of BaP is extraordinarily com-
plex, and alterations in one of the many
pathways could affect BaP-induced tumor
. · ~ . . . . .
Genesis. l ile lnltla OX1C .atlon to arene
oxides is controlled by the cytochrome
P-450 system. The existence of cytochrome
P-450 in multiple forms that differ in
their capacity to catalyze oxidation of BaP
at different positions is well established
(Coon 1981; Conney 1982; Gelboin 1983~.
The distribution of these forms is depen-
dent on the tissue of interest, the strain
and species, and to a great extent on the
effects of numerous inducers. Inducibility
can be controlled by genetic or environ-
mental factors. Thus it is clear that the
production of particular arene oxides and
phenolic metabolites of BaP in a given
system will depend on a great many con-
tributing factors.
The fate of the arene oxide intermediates
is controlled to a large extent by the en
OCR for page 565
Stephen S. Hecht
OH
HO'
Oh
1 1'~
HO i" tetraolS
"~( HO'''˘(: glutathione
OH OH conjugates
anti-7,8-diol- syn-7,8-diol
9,10-epoxide 9, 10-epoxide
1
Ah/
565
e ( 8 ) _ ( 2 2
8 e ( ) 6 ( ) OH )
. ~O ~OH 1 ~
1 1~
- glutathione
conjugates
)~:
OH \* glucuronides
I DNA
dg
NH
H O,,:
of
OH
1 ,6-quinone
3,6-quinone
6, 1 2-quinone
Figure 2. Metabolism of benzota]pyrene. (Adapted from LaVoie and Hecht 1981.)
sulfates
glucuronides
zyme epoxide hydrolase. Its tissue concen- BHA, taken together with related studies,
"rations depend on species, inducer pre
treatment, and on the presence of inhibitors
such as 1, 1,1-trichloropropene oxide
(Oesch 1980~. Conjugation of phenolic and
dihydrodiol metabolites catalyzed by gluc
uronyl transferases, and detoxification of
epoxides and dihydrodiol epoxides by mul
tiple forms of glutathione-S-transferases
are, like the cytochrome P-450 and epoxide
hydrolase activities, dependent on multiple
factors.
The effects on metabolic activation and
detoxification pathways of modifiers of
BaP tumorigenesis are complex, but they
do provide insights on potential mecha
nisms of cocarcinogenesis or inhibition.
Studies of the cocarcinogens catechol, fluo
ranthene, pyrene, benzo~e~pyrene, and
are for the most part consistent with the
concept that formation of 7,8-diol-9,10-
epoxides is an important activation path-
way in BaP-induced tumorigenesis.
Metabolism in Human Tissues. The me-
tabolism of BaP has been extensively stud-
ied in various subcellular, cellular, and or-
gan culture systems from human tissues
(International Agency for Research on
Cancer 1983~. These studies have shown
that the basic pattern of BaP metabolism is
qualitatively similar in human tissues and in
laboratory animal tissues, but major quan-
titative differences can occur. The forma-
tion of DNA adducts via the 7,8-diol-9,10-
epoxide pathway is regularly observed in
human tissues.
OCR for page 568
568
Effects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
vitro is 1,2-dihydro-1,2-dihydroxy-6-ni-
trochrysene. Formation of the correspond-
ing 7,8-dihydrodiol is not observed, indi-
cating that the 6-nitro group inhibits
oxidation at the adjacent position (El-Ba-
youmy and Hecht 1984b). Inhibition of
dihydrodiol formation has similarly been
observed in studies of 6-nitrobenzo~aipy-
rene, although an 8,9-dihydrodiol has been
detected as a metabolite of 7-nitrobenz-
[aJanthracene (Fu et al. 1982; Fu and Yang
1983~. Nitro-reduction alone does not gen-
erally appear to be a major metabolic path-
way for either 6-nitrochrysene or 6-nitro-
benzota~pyrene. These results contrast to
those observed with 1-nitropyrene (El-Ba-
youmy and Hecht 1983, 1984b), and with
the dinitropyrenes that appear to be acti-
vated at least partially by nitro-reduction
followed by O-acetylation (Beland et al.
1985~.
Our present knowledge of the mecha-
nisms of nitro-PAH metabolic activation is
inadequate. The general aspects of activa-
tion and detoxification are not clearly un-
derstood, even for 1-nitropyrene which is
the most extensively studied nitro-PAH.
Further research is needed in this area.
Given the advanced state of technology and
experience with other carcinogens, these
studies should proceed rapidly.
Research Problems Relating to
the Potential Carcinogenic
Effects of Polyaromatic
Hydrocarbons and Nitro-
Polyaromatic Hydrocarbons
in Humans
Individual Dosimetry
The wide interindividual variation in PAH
metabolism observed in studies on human
tissue samples or cells is a result of the
extraordinary complexity of the metabolic
pathways and the many factors, genetic as
well as environmental, that control the
levels of the various enzyme activities in-
volved. DNA adduct formation and pro-
tein adduct formation are significant end
points of these processes because they re-
flect the generation of electrophilic inter
mediates in PAH metabolism, and the
persistence of PAM/DNA adducts in repli-
cating cells is probably one important de
. . . . . · · · -
termlnant tor 1nltlatlon ot carclnogenesls.
Although extensive studies of DNA adduct
and protein adduct formation and persis-
tence after single administrations of various
doses of PAHs have been performed, little
if any information is available concerning
these end points under conditions of
chronic PAH treatment (Stowers and An-
derson 1985~.
It will be essential to perform such stud-
ies in animals treated with doses of PAHs
known to result in differing tumor inci-
dences and to determine the relationship, if
any,. between DNA adduct and protein
adduct levels and tumor development.
These chronic administration experiments
are important because they are more closely
related to conditions of human exposure
than are the acute administration protocols.
The availability of sensitive assays for
DNA adducts and protein adducts, without
using labeled PAHs, will make these
chronic studies feasible. The results of these
investigations will be important in forming
a baseline for interpretation of analogous
data obtained from measurements of DNA
adducts and protein adducts in humans.
· Recommendation 1. The structures of
the major DNA adducts and protein ad-
ducts formed from representative PAHs
and nitro-PAHs should be determined in
laboratory animals.
For dosimetry studies to be undertaken,
the structures of appropriate DNA adducts
and protein adducts must be known. These
studies should initially focus on repre-
sentative PAHs and nitro-PAHs: BaP,
benzo~blfluoranthene, fluoranthene, 1-ni-
tropyrene, 1,6-dinitropyrene, and 6-nitro-
chrysene are recommended based on
current knowledge of their environmental
occurrence and carcinogenicity in labora-
tory animals. This list could change as
further data become available. Among
these six compounds, the structures of ma-
jor DNA adducts formed in vivo are
known only for BaP. Blood protein ad-
ducts are formed via BaP-7,8-diol-9,10
OCR for page 569
Stephen S. Hecht
569
epoxide (Santella et al. 1986; Shugart 1986~.
Further studies are required to characterize
the DNA adducts and blood protein ad-
ducts of the other five compounds.
~ Recommendation 2. Methods should
be developed for determining individual
uptake and metabolic activation of repre-
sentative PAHs and nitro-PAHs.
Although our understanding of the proc-
esses involved in tumor development is
incomplete, there is no question that the
metabolic generation of specific reactive
PAH or nitro-PAH metabolites is one im-
portant feature of the process. These
metabolites bind to DNA and protein. The
measurement of DNA adducts provides a
biologically significant end point which
bypasses the many variables involved in
individual exposure to, uptake of, and me-
tabolism of PAHs. A drawback of DNA
adducts as dosimeters is that they are re-
moved from various cells at different rates
depending on repair mechanisms and on
normal cellular turnover.
In contrast, adducts with proteins such as
hemoglobin have a more predictable and
longer lifetime. The lifetime of hemoglobin
in humans is four months and thus PAH/
hemoglobin adducts could provide a mea-
sure of chronic exposure (Calleman et al.
1978; Garner 1985~. Although such adducts
may not be biologically significant per se,
they do provide a cumulative measure of
individual exposure to PAHs and activation
of PAHs to electrophiles. Thus, methods
should be developed to measure DNA ad-
ducts and protein adducts of the six com-
pounds listed above.
~ ~ ~ 1 '1 1 1 ~
Several methods are available tor sens~-
tive detection of PAM/DNA adducts. Im-
munoassay techniques have been developed
and applied to the analyses of BaP/DNA
adducts in human tissue samples and in
peripheral blood lymphocytes (Harris et al.
1985; Santella et al. 1985~. These studies
have indicated the presence of these adducts
in lung cancer patients, coke oven workers,
roofers, and foundry workers, but not in
noncancer patients.
Other methods for measuring DNA ad-
ducts include synchronous fluorescence
spectrophotometry (Harris et al. 1985) and
fluorescence-line-narrowed spectra (Heisig
et al. 1984~. An alternative method that
shows great promise for a variety of
PAM/DNA adducts is the 32P-postlabeling
technique (Randerath et al. 1985~. Al-
though some of these techniques are still in
the developmental stage and require refine-
ment before being applied routinely to a
variety of representative PAHs and nitro-
PAHs, they are generally promising.
Methods for assessing formation of
PAH/protein adducts are being developed
(Santella et al. 1986; Shugart 1986~. On the
basis of results obtained with taminobi-
phenyl (Green et al. 1984), the measure-
ment of nitro-PAH/hemoglobin adducts is
likely to be feasible. The further develop-
ment of these methods should certainly be
a focus of future research.
In addition to the measurement of
PAM/DNA adducts and PAH/protein ad-
ducts, there are a number of other methods
for assessing carcinogen activation which
may be appropriate as an adjunct to the
approaches described above. These include
the measurement of urinary or fecal metab-
olites (Becher and Bjorseth 1983), the use
of monoclonal antibodies to type human
tissues for individual cytochrome P-450s
(Gelboin 1983), the detection of antibodies
to PAM/DNA adducts (Harris et al. 1985),
and the use of blood cells to metabolically
activate BaP in vitro (Gelboin 1983~.
Recommendation 3. Under condi-
tions of chronic administration of PAHs or
nitro-PAHs to laboratory animals, the re-
lationship between DNA adduct or protein
adduct formation and tumor development
should be determined.
In order to assess the relationship of PAH
or nitro-PAH adducts with DNA or pro-
tein to the risk for cancer development, it
will be necessary to perform chronic stud-
ies in laboratory animals. The most desir-
able route of administration for such stud-
ies would be inhalation, but practical
considerations prohibit extensive use of this
model. As a compromise, concurrent use of
the Syrian golden hamster intratracheal in-
stillation model is recommended. Groups of
OCR for page 570
570
Effects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
hamsters should be treated chronically with
a range of doses of the six representative
compounds listed above with Recommen-
dation 1, as well as mixtures of these com-
pounds.
DNA adducts should be measured in
respiratory tract tissue, in cheek pouch or
other nonrespiratory tissues, and in periph-
eral blood lymphocytes. Blood protein ad-
ducts should also be quantified. Parallel
inhalation studies with selected compounds
should be carried out to validate the results
obtained by intratracheal instillation. The
results should be evaluated in light of the
tumor incidence in the various groups.
These data will provide the basis for inter-
preting data that eventually will be ob-
tained from human populations potentially
exposed to PAHs and nitro-PAHs.
These measurements in humans, taken
together with the data from laboratory
animals, should allow determination of in-
dividual PAH dose and may provide mark-
ers for assessing individual risk for cancer
development. Such markers would be in-
valuable in epidemiologic studies. The lack
of data relating levels of DNA adducts and
protein adducts to cancer risk in laboratory
animals, under conditions of chronic PAH
or nitro-PAH treatment, is probably the
most significant gap in our present ability
to assess risk in humans, given the fact that
methods for making these measurements
are becoming available.
Recommendation 4. Pilot studies
should be undertaken on individuals poten-
tially exposed to PAHs or nitro-PAHs in
order to determine the feasibility of moni-
toring DNA adducts and protein adducts in
humans.
Application to humans is the goal of
developing methods for measuring individ-
ual dosimetry of PAHs and nitro-PAHs.
Therefore, it is essential that the feasibility
of these methods be assessed. As mentioned
above, studies of this type are already ongo-
ing for some PAM/DNA adducts and aro-
matic amine/protein adducts. When assays
have been shown in animal studies to have
the requisite sensitivity, they should be ap-
plied to groups of 2~50 individuals who are
supposed to have been exposed to PAHs or
nitro-PAHs, and to corresponding numbers
of controls. The results should be carefully
cross-checked with other methods, to ensure
their validity. For example, PAM/DNA ad-
ducts could be measured by immunoassay
and by 32P-postlabeling. If both methods are
valid, the results should agree. Multilabora-
tory collaborative studies should be under-
taken for cross checking of results. Parallel
assays should also be made to aid in identifi-
cation of the exposure source, for example,
. . . . . .
urinary or sa Vary nicotine or cot~mne as
monitors for tobacco exposure.
Bioassays in Laboratory Animals
Although extensive evaluations of PAH
tumorigenicity have been performed using
the mouse skin bioassay system, and struc-
ture/activity relationships are fairly well
understood, limited data are available on
induction of respiratory tumors by PAHs.
Assays by intratracheal instillation have
been performed with only a few PAHs that
are found in mobile source emissions (Sel-
lakumar and Shubik 1974~. Several other
PAHs have been tested by lung implanta-
tion with results not entirely in agreement
with expectations based on mouse skin
studies Stanton et al. 1972; Deutsch-
Wenzel et al. 1983~. Inhalation experiments
are practically nonexistent. It is possible
that the extensive reliance on mouse skin
assays could give a distorted perspective of
the potential importance of particular
PAHs in respiratory carcinogenesis.
Recommendation 5. Inhalation bioas-
says and intratracheal instillation bioassays
of selected PAHs should be performed.
Although inhalation experiments on
eAiis are necessary, practical consider-
ations demand that they be somewhat lim-
ited. Of the PAHs present in mobile source
emissions, BaP and benzo~b]fluoranthene
are the most tumorigenic on mouse skin.
They are also tumorigenic in the rat lung
implantation system. It would be impor-
tant to determine the comparative carcino-
genicity of these two hydrocarbons by in-
halation studies in rats or Syrian golden
OCR for page 571
Stephen S. Hecht
571
hamsters. Fluoranthene and pyrene are the
two most prevalent cocarcinogenic hydro-
carbons in mobile exhaust emissions, ac-
cording to mouse skin assays. Their cocar-
cinogenicity with BaP in inhalation assays
should be tested.
Intratracheal instillation bioassays have
been performed on only a limited num-
ber of PAHs present in mobile source
emissions. These studies should be ex-
tended at least to some of the more preva-
lent or carcinogenic components such
as anthanthrene, benzo~ghi]fluoranthene,
benzo ~ ~ fluoranthene, benzo [ah i] perylene,
5-methylchrysene, cyclopentatcd~pyrene,
fluoranthene, indeno[1,2,3-cdipyrene, and
pyrene. The intratracheal instillation model
would probably also be the most suitable
for testing mixtures of PAHs as they occur
. .. . .
In mo ~1. .e source emissions.
Bioassays of nitro-PAHs have been lim-
ited, and structure/activity relationships are
not yet predictable. Extensive systematic
studies on nitro-PAH carcinogenicity in
several model systems are required. In ad-
dition, it is possible that carcinogenic activ-
ities of PAH mixtures could differ signifi-
cantly from expectations based solely on
activities of the components of the mix-
tures. Many examples of cocarcinogenic or
inhibitory activities of one PAH upon an-
other are known. It would be important to
assess the respiratory carcinogenicity of
PAH/nitro-PAH mixtures, using relative
concentrations similar to those observed in
mobile source emissions.
· Recommendation 6. The tumorige-
nicity of environmental nitro-PAHs should
be evaluated.
percents to complement those already in
progress with 1-nitropyrene.
Limited data are available on the effects
of modifiers of PAH carcinogenesis in res-
piratory tract carcinogenesis models. It
would be important to determine the ef-
fects of such cocarcinogens as catechol, or
of dietary inhibitors, on PAH carcinogen-
esis in the respiratory tract. In addition,
only limited data are available on the po-
tential cocarcinogenicity and tumor-pro-
moting activities of compounds to which
humans are extensively exposed.
~ Recommendation 7. Bioassays should
be performed to discover environmental
modifiers of PAH and nitro-PAH carcino
. .
genlclty.
To study environmental modifiers of
PAH and nitro-PAH carcinogenicity, rela-
tively inexpensive assay systems such as
mouse skin, mouse forestomach, A/}
mouse lung, or the newborn mouse should
be used, with BaP, benzo~b]fluoranthene,
fluoranthene, 1-nitropyrene, 1, 6-dinitro-
pyrene, and 6-nitrochrysene as repre-
sentative carcinogens. This work should
focus on exploring structural analogues of
known cocarcinogens, such as catechol, or
analogues of known chemopreventive
agents such as p-methoxyphenol, indole-3-
carbinol, benzyl isothiocyanate, or sodium
selenite and related organoselenium com-
pounds. Emphasis should be on those com-
pounds to which humans are exposed in
relatively high concentrations.
The most appropriate system for bioas-
says of nitro-PAHs is probably intratra-
cheal instillation, at least for screening the
activities of the numerous nitro-PAHs that
are present in diesel exhaust. Studies with
1,6-dinitropyrene have shown that it does
induce lung tumors when applied by intra-
tracheal instillation in hamsters (Takayama
et al. 1985~. The nitro-PAH showing the
~reatest activity in other model systems,
such as 1,6-dinitropyrene and 6-nitrochry-
sene, should be chosen for inhalation ex
Mechanisms of Polyaromatic Hydrocarbon
and Nitro-Polyaromatic Hydrocarbon
Ca rcin ogen es is
Although a great deal is known about the
metabolic activation of BaP as a repre-
sentative PAH, there are many unanswered
questions about the process by which ex-
posure to BaP results in tumor induction. It
is well established that BaP must undergo
enzymatic oxidation to an electrophile that
can react with DNA, and that DNA is the
key macromolecular target for initiating
the tumorigenic process. It is also known
that a BaP-7,8-diol-9,10-epoxide is one of
OCR for page 572
572
Effects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
the major DNA binding metabolites. How-
ever, multiple DNA adducts are formed
from BaP and it is not known which of
these, or which combination of adducts, is
most important in tumor initiation. Other
aspects of cellular damage by BaP that may
enhance the effects of DNA damage, such
as free-radical generation, and their rela-
tionship to tumor initiation, remain largely
unexplored at present.
The steps by which modified DNA can
cause transformation of cells resulting even-
tually in frank appearance of tumors remain
poorly understood, although it is known that
BaP-7,8-diol-9,10-epoxide can activate the
c-Ha-ras-1 oncogene (Marshall et al. 1984~.
The role of oncogenes in carcinogenesis is an
exciting area of investigation which may
provide major leads in understanding the
process of tumor development. The effects of
BaP metabolites, or of endogenous factors,
as cocarcinogens or tumor promoters in BaP
carcinogenesis need to be explored.
Whatever gaps exist in our knowledge
about BaP carcinogenesis are even greater
for most other PAHs. With the possible
exception of 7,12-dimethylbenz~ajanthra-
cene (DMBA), no PAH has been so exten-
sively investigated as BaP. Important dif-
ferences are found in the mechanisms of
activation of BaP and DMBA (ripple et al.
1984~. Therefore, further research is needed
on the mechanisms of tumor induction by
other major carcinogenic PAHs found in
mobile source emissions, in particular the
benzofluoranthenes.
· Recommendation 8. The mechanisms
by which carcinogenic PAHs and nitro-
PAHs undergo metabolic activation and
detoxification should be determined.
Rational evaluation of the potential car-
cinogenic effects of PAHs and nitro-PAHs
in humans requires a basic understanding of
the major pathways of metabolic activation
and detoxification of these compounds in
laboratory animals. The most important
tumorigenic compounds that require fur-
ther study for elucidation of these basic
pathways include the six listed with Rec-
ommendation 1 as well as benzot]fluoran-
thene, indenot1,2,3-cdipyrene, 1,3-dinitro-
pyrene, and 1,8-dinitropyrene.
These studies should be performed in the
species and tissues in which these com-
pounds induce tumors. In general, in vitro
experiments are useful for identification of
metabolites and evaluation of their role in
metabolic activation. However, in vivo
studies are essential for relating metabolic
pathways to carcinogenesis. In particular,
. . . .
extensive furt ner investigations are neces-
sary on the disposition, metabolism, and
DNA binding of PAHs and nitro-PAHs
under conditions of inhalation exposure.
Although modifiers of carcinogenesis are
important in determining whether or not a
PAH or nitro-PAH will induce cancer,
research on the mechanisms by which they
affect the carcinogenic process is still fairly
limited. The studies carried out so far indi-
cate a complex network of effects. It will be
important to select one or two environ-
mentally prevalent modifiers of PAH car-
cinogenesis and to carry out in-depth inves-
tigations of their mechanisms of action.
~ Recommendation 9. The mechanisms
by which environmental compounds mod-
ify PAH and nitro-PAH carcinogenicity
should be investigated.
Further in vitro and in vivo studies
should probe the mechanistic basis for the
cocarcinogenic effects of such compounds
as fluoranthene, pyrene, and catechol and
for the inhibitory effects of certain phenols,
isothiocyanates, and indoles. Since these
effects can be extraordinarily complex, it is
recommended that the studies focus on one
or two cocarcinogens, such as fluoranthene
and catechol, and one or two appropriate
inhibitors such as p-methoxyphenol and
indole-3-acetonitrile. These studies should
be performed initially with BaP in mice
because of the extensive data base that
. . .
ex~sts on th~s system.
Summary
Epidemiologic studies have been per-
formed on cohorts exposed to numerous
mixtures containing PAHs, including to-
bacco smoke, coal tars, and soots. Many of
these studies have shown that exposure to
OCR for page 573
Stephen S. Hecht
573
such mixtures causes cancer at various tis
sue sites, but individual PAHs have not
been specifically incriminated as the caus
ative agents because they occur together
with other carcinogens. Nevertheless, these
data, taken together with the extensive an
imal bioassays of PAHs, strongly indicate
that PAHs can cause cancer in humans. The
role of nitro-PAHs in human cancer is
unclear at present.
Studies of PAH metabolism in animal
and human tissues have elucidated many of
the important pathways of activation present.
and detoxification and have demonstrated
that their metabolism is extremely com-
plex. The balance of activation versus
detoxification can be influenced by a mul-
titude of genetic and environmental fac-
tors. However, it is clear that formation
of specific PAH metabolite/DNA adducts
is the key step in the initiation of the
carcinogenic process. Similar conclusions
can be drawn about nitro-PAHs, but their
metabolic activation and detoxification
pathways are not very well characterized at
Summary of Research Recommendations
HIGH PRIORITY
Recommendation 1 The structures of the major DNA adducts and protein adducts
formed from representative PAHs and nitro-PAHs should be
determined in laboratory animals.
Recommendation 2 Methods should be developed for determining individual uptake
and metabolic activation of representative PAHs and nitro-PAHs.
Recommendation 3 Under conditions of chronic administration of PAHs or nitro
PAHs to laboratory animals, the relationship between DNA adduct
or protein adduct formation and tumor development should be
determined.
Recommendation4 Pilot studies should be undertaken on individuals potentially
exposed to PAHs or nitro-PAHs in order to determine the feasi
bility of monitoring DNA adducts and protein adducts in humans.
Recommendation 8 The mechanisms by which carcinogenic PAHs and nitro-PAHs
undergo metabolic activation and detoxification should be deter
mined.
MEDIUM PRIORITY
Recommendations Inhalation bioassays and intratracheal instillation bioassays of
selected PAHs should be performed.
Recommendations The tumorigenicity of environmental nitro-PAHs should be
evaluated.
LOW PRIORITY
Recommendation 7 Bioassays should be performed to discover environmental mod
ifiers of PAH and nitro-PAH carcinogenicity.
Recommendation 9 The mechanisms by which environmental compounds modify
PAH and nitro-PAH carcinogenicity should be investigated.
OCR for page 574
574
Effects of Polynuclear Aromatic Hydrocarbons and Nitroaromatics
Acknowledgments
I thank Ms. Gail Thiede for typing and
editing the manuscript. Our studies on
PAHs and nitro-PAHs are supported by
NCI Grants CA-44377 and CA-35519.
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Representative terms from entire chapter:
dna adducts
