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OCR for page 145
for mutagenic activity. 30, 132, 167, 168, 171 Wang et al. 177
collected air samples from a res idential area at an intersection of two
heavily trafficked crossroads in the Buffalo, New York, area.
Extraction of the particulate fraction with acetone resulted in a
preparation highly mutagenic in Salmonella strains TA 98, TA 100, and
TA 1537. These investigators also obtained a positive direct mutagenic
response with automobile-exhaust samples from a spark-ignition
internal-combus Lion engine (with leaded gas as the fuel). The
mutagenic ingredients appeared to originate in motor oil during the
combustion process and were not due to lead. Similar results have been
obtained by Pitts et al.132 with atmospheric particulate extracts
from the Los Angeles basin, by Teranishi et al. , 168 by Tokiwa et
al. 171 with extracts from several Japanese cities, and by Talcott and
Weil67 and Commoner et al.30 with extracts from other American
cities. Unfortunately, the quantitation of some of these studies may
be open to question because of filter artifacts. The disposition of
the filter apparatus in relation to sunlight, temperature, etc., is
important, because these factors may facilitate chemical reactions
involving PAHs and may result in artifactual formation of mutagens.
This aspect is discussed in Chapter 3.
Soot makes up 2-15: of the mass of fine particles that are present
in urban atmospheres.105 A number of studies have been conducted to
establish its mutagenic potential. Kadin et al .83 have experimen-
tally generated soot from ingredients with varied sulfur composition
--i.e., from pyridine, decalin, and o-xylene or from th~iophene,
decalin, and o-xylene--and have compared its mutagenicity with that of
soot obtained from burned kerosene. Dichloromethane extracts of all
the soots were mutagenic in a bacterial assay in which a forward muta-
tion of 8-azaguanine resistance was measured. The soots generated from
the sulfur-containing and nitrogen-containing ingredients, as well as
soots from kerosene or furnace black, exhibited 10-17: of the mutagenic
activity of authentic BaP (on a weight basis ~ .
Emission from spark-ignition combustion and diesel engines has been
tested for mutagenic activity in the Salmonella system.27~79~83~10l
It is known that particulate emission from light-duty diesel engines is
considerably greater than that from light-duty catalyst-equi~ped
spark-ignition engines--i.e., 0.2-1.0 vs. 0.006-0.02 g/mi.l4 Table
4-8 presents data of Claxton 8 relative to comparative mutagenic
activity of emission of diesel and spark-ignition engines, of
cigarette-smoke condensate, of coke-oven emission, of roofing-tar
emission, and of BaP (positive control). The results are reported in
terms of revertants/100 fig of soluble dichloromethane organic
compounds; the soluble organic components represent approximately 25%
of the total mass of the particles. As is evident from the table,
cigarette-smoke condensate, roofing tar, and BaP required metabolic
activation by an S-9 fraction, whereas diesel-engine exhaust was
directly mutagenic. The other kinds of emission were both directly and
indirectly mutagenic. The diesel exhaust exhibited a wide range of
mutagenic activity, although the high value is probably peculiar to the
4-11
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particular engine that generated the emission.
far greater than that of emission.
The activity of BaP is
Naman and Clarkll9 have determined the quantity of particles
emitted and the mutagenic activity of extracts of the exhausts of
several spark-ignition engines that burned gasoline, a 901/10% ethanol
blend, or commercial gasohol. The results are presented in Table 4-9.
Although the number of rever ten ts per mile differed for each of the
four-cylinder engines, the addition of ethanol clearly reduced the
direct mutagenic capacity.
Several investigators have determined the mutagenic activity of
respirable coal fly ash,25~69 which does have mutagenic activity in
the Salmonella/microsome assay. Virtually all the emissions yield a
mutagenic response in this test system.
Extracts from the various kinds of emission contain a large number
of PAHs, among which is BaP.48~89~176 Extracts of diesel particles
have been separated on Sephadex LH-20 into six fractions;61 the
contribution of each to the total mass of the diesel extract obtained
from a low-sulfur and high-sulfur fuel is shown in Table 4-10.
Furthermore, each of these exhausts was obtained before or after
passage through an oxidative catalyst. Fraction 1 contained most of
the mass of the extracts from both fuel exhausts. However, fractions 3
and 4 contained most of the mutagenic activity. Fraction 3 from the
low-sulfur exhaust contained the bulk of the PAHs, including
phenanthrene, methylphenanthrenes, fluoranthene, pyrene, methylpyrenes,
benzo[ghi]fluoranthene, benzanthracene (BA), chrysene (or
benzo[c]phenanthrene), methyl-BAs, and BeP (or perylene).61 With the
high-sulfur fuel, one found, in addition, the methylbenzothiophenes.
It is of interest that the low-sulfur fuel gave an exhaust whose
mutagenic activity was increased after passage through a catalyst. The
reverse was true for the high-sulfur fuel. Furthermore, fraction 4
from the high-sulfur fuel, before oxidative catalysis, proved the most
mutagenic.
The major identified components of emission have been tested for
mutagenic activity with the Salmonella forward-mutation assay of Thilly
and co-workers.83~10l In this assay, mutants that are resistant to
the purine analogue 8-azaguanine are scored. Of the components present
in kerosene-soot extract, cyclopenta[cd]pyrene proved the most
mutagenic; it was also present in the highest concentration (see Table
4-11~. C~cloyentatcd~pyrene is a known component of all
soots, 53' 74' 75 of cigarette smoke,l59 of automobile exhaust,57
and of coal fly ash.25 The sum of the mutagenicities of the
identified individual PAHs was slightly greater than that of the
kerosene-soot extract itself. The total mutagenic activity of the
kerosene-soot extract could almost be reproduced by that of the
cyc lopenta ~ cd ~ pyrene .
The investigators compared the mutagenic efficacy of additional
PAHs with and without an S-9 preparation, using induced cells from the
liver; the results are in Table 4-12. Methylation of several of the
4-12
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inactive PAHs, such as anthracene and phenanthrene, resulted in the
acquisition of mutagenicity. Preliminary evidence has led the Thilly
group to suspect the presence of alkyl-substituted anthracene and
phenanthry~ in diesel-soot fractions that were mutagenic in
bacteria. In this series, the most active compound was perylene,
which was followed by cyclopenta [cd] pyrene. The mutagenicity of
cyclopenta ~cd] pyrene in the Salmonglla/microsome assay has been
confirmed by Eisenstadt and Gold;4 metabolic activation by the S-9
fraction was required before this mutagenic property was elicited.
Ni treated PARE
As mentioned previously, emission from either diesel or
spark-ignition engines exhibits considerable direct-acting mutagenic
activity in the Salmonella/microsome assay, whereas cigarette-smoke
condensates, roofing-tar extracts, and BaP do not. This has led
several investigators to study engine exhaust for the presence of
direct-acting PAR derivatives that might have been produced by gaseous
exhaust components--~.g., nitrogen oxides--or by atmospheric oxidative
reactions involving ozone. Various nitropyrenes and other analogues
have been assayed for mutagenic activity in the bacterial
system 52,96,111,112,124,130 These substances exhibit potent
activity in the Salmonella mutagenesis assay. Indeed, it has been
estimated by Gorse (personal communication) that the concentration of
nitropyrene alone in diesel particulate extracts could account for
13-24: of the total direct mutagenic activity with-TA 98. Tokiwa et
al.170 have assayed the mutagenicity of the nitrophenanthrenes,
1-nitropyrene, 3-nitrofluoranthene, and 6-nitrochrysene. Each of the
parent PAHs was inactive as a direct mutagen, but 6-nitrochrysene was
slightly active, nitrophenanthrene was active, and 1-nitropyrene was
most active against TA 98 and TA 100. S-9 was not required for this
demonstration of mutagenic activity. Pitts et al .132 reported the
direct mutagenic activity of 1-, 3-, and 6-nttrobenzo~aipyrene in the
Salmonella/microsome assay. Perylene, another exhaust constituent that
is converted to 3-nitroperylene, demonstrated mutagenesis.132 In a
similar fashion, nitrated derivatives of anthracene, fluoranthene,
benz~aJanthracene, benzo~kifluoranthene, and benzotghi~perylene--all of
which are present in diesel exhaus t--exhibited potent mutagenic
activity in the Salmonella assay.16
The nitropyrenes have been reported as contaminants of xerographic
copiers and toners which may therefore contribute to the problem of
mutagenicity. 103 ~ 143 Rosenkranz et al . 143 have demonstrated the
presence of such a mutagenic activity with various Salmonella test
s trains; they have traced this property to nitropyrenes that were
present as impurities in carbon black. In addition to mononitrated
components, they were able to identify the 1,3-, 1,6-, and
1,8-dinitropyrenes, 1,3,6-trinitropyrene, and 1,3,6,8-tetranitropyrene
as contaminants. All these derivatives demonstrated direct mutagenic
activity (see Table 4-13) with both nitroreductase-positive and
-negative variants of Salmonella. The mutagenic property of
4-13
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1-nitropyrene and 1,3-dinitropyrene depended heavily on the endogenous
bacterial nitroreductase activity. Insertion of two nitro groups in
the pyrene moiety increased mutagenic activity by a factor of approxi-
mately 100, although information is ins uf ficient to extrapolate to
other PAHs. The most potent of these derivatives was 1,8-dinitro-
pyrene. It was striking that, of the three dinitro derivatives, two
ac t ed independent ly 0 f endogenous n i troreduc base; equa 1 numbers 0 f
revertants per nanomole are observed in both TA 98 and TA 98 NR. A
similar situation occurred with the trinitropyrenes and tetranitro-
pyrene. The mutagenic activity of 1,8-dinitropyrene is the highest
ever recorded in the 1 iterature, 112 The presence of the 1, 6- and
1,8-dinitropyrenes as predominant mutagenic components in diesel-
particle extract has been confirmed by Pederson and Siak,124 who
estimated that 15-20: of the total mutagenic activity of the extract
may be contributed by these dinitropyrenes (in addition to as much as
24t contributed by 1-nitropyrene).
Sulfur-Containin~,PAHs
The presence of sulfur-containing heterocyclic PAHs has been
reported in various combustion products, particularly from high-sulfur
petroleum products (see Chapter 1). In many heterocyclic structures,
one aromatic ring has been replaced by thiophene.86 It is anti-
cipated that the increase in the use of coal, particularly with high
sulfur content, will result in substantial environmental pollution with
these ingredients . Thus, it is imperative to have a bet ter understand-
ing of the biologic effects of these sulfur-containing heterocyclic
PAHs.
The mutagenic ity of several s ul fur-containing PAHs has been
determined in the Salmonella/microsome assay by Karcher et al.,87 and
the results are presented in Table 4-14 . Of the isomers listed,
benzo[2,3Jphenanthro[4,5-bcd] thiophene was the most potent; its ring
configuration corresponds to that of BaP, although it is more mutagenic
than the la t ter .
ANIMAL-CELL MUTAG~.N~.~ T ~
A number of animal-cell model systems have been used to ascertain
the mutagenic effects of combustion-engine emission, as well as other
exhaul7' 1 ~ ese have been reviewed in previous monographs on
PAHs. ~ Many of the tests depend on the selection of variants
on the basis of resistance to 8-azaguanine, 6-thioguanine, ouabain, or
deoxythymidine analogues.
Comparative data on the development of 6-thioguanine resistance in
Chin22e hamster ovary (CHO) cells have been reported by Casto et
al., who used extracts of diesel-exhaust particles and coke-oven
emission (see Table 4-15~. All extracts yielded the same number of
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mutant cells, which was comparable with that of the positive control,
methyl methanesulfonate, a known direct-acting methylating agent.
Curren et al.34 have tested the production of ouabain-resistant
BALB/c 3T3 cells when the latter were exposed to a variety of agents
(see Table 4-16~. The spark-ignition-engine extract was considerably
more mutagenic in this assay than the diesel extracts. A roofing-tar
pot sample and coke-oven emission also exhibited greater mutagenic
efficacy. The presence of an activating system did not significantly
affect mutagenicity. The extract from the gasoline-engine exhaust
appeared more mutagenic than the extracts from various diesel engines,
and coke-oven pot samples were even more active. Mutability at several
different genetic loci by PAHs has been determined by Huberman and
Sachs78 (Table 4-17~. Good mutagenic activity with respect to the
HGPRT locus was manifested by dibenz~acianthracene,
dibenz~ah~anthracene, 7-methylbenzanthracene, BaP, 7,12-DMBA, and
3-MC. The last four compounds named were also mutagenic with respect
to ouabain resistance. At both loci, 7,12-DMBA was most active.
As indicated previously, diesel exhaust demonstrates considerable
direct mutagenic activity in the Salmonella/microsome assay. The
nitro-PAHs have been considered as likely candidates for this
activity. Thilly and colleaguesl°l have been unable to demonstrate
any direct mutagenic activity with human lymphoblasts as the target
cells, although, in the presence of an activating system, a consider-
able amount of 6-thioguanine resistance and trifluorothymidine
resistance resulted after addition of diesel extract to the culture
media. These experiments suggest that the nitrated PAHs, if present in
the diese1 extracts, are rapidly inactivated by the lymphoblasts or
require for activation a nitroreductase (or other enzyme) that is
absent from these cells. Indeed, application of the term "direct-
acting mutagen" to the nitrated PAHs is not entirely correct. It is
postulated that these analogues undergo a reduction, catalyzed by a
nitroreductase, to an amino derivative that may be further transformed
into reactive hydroxylamino PAHs (see Chapter 3~. The latter would
easily form electrophilic substances that could interact with DNA in
causing a mutation. What is needed is additional experimentation on
the mechanism of action of the nitrated PAHs in both bacterial and
mammalian-cell systems.
Sister chromatic exchange has been used to assess genotoxic
activity of various kinds of emission. Unfortunately, SCE appears to
be more predictive of point mutation than of frameshift mutation,l9
whereas most of the PAHs produce the latter damage. The experiments of
Mitchell et al ., 116 which used CHO cells, indicated that all the
emission extracts were inferior to BaP in inducing SCE. Of the emis-
sions, coke-oven extracts proved the most active, and the heavy-duty
Caterpillar dresel-engine exhaust was the least potent. Intermediate in
activity were cigarette-smoke condensate, roofing-tar emission, Mustang
gasoline-engine emission, and other diesel-engine emission. None of
these required metabolic activation for SCE activity.
4-15
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The induction of SCE has been performed in viva with Chinese
hamsters that were given various PAHs intraperitoneally.1 After
two injections, the bone marrow was aspirated, and the SCEs per
metaphase cell were determined. Although the positive control, BaP,
did produce SCE, there was little correlation between the quantitative
aspects and the carcinogenic potential of the PAHs. No comparable
experiments were performed with the various kinds of emission. The
experiments of Schonwald et al.150 also showed a lack of correlation
between carcinogenicity and ~E. These investigators determined SCE
induced by BaP with human lymphocytes obtained from normal persons and
lung-cancer patients; no difference was observed. Guerrero et al.58a
intratracheally exposed Syrian hamsters to 200 ng of BaP over a 10-wk
period, examined in vitro cultures of lung tissue for sister chromatic
exchange (SCE), and concluded from the results that BaP was
metabolically activated by lung cells in viva. In other studies,
diesel exhaust particles (DEP) in doses of 0-20 mg per hamster were
administered over a 24-h period; although the study was limited in
scope, the results demonstrated that DEP can induce genotoxic damage.
CARC INOGENE SIS
SKIN
Kotin and colleagues89~91 first reported the presence of carcino-
genic substances in the exhaust of gasoline and diesel engines. Benzene
extracts of particles from these sources produced both papillomas and
carcinomas when applied to the skin of mice. These studies were
extended by Wynder and Hoffmann,l82 who compared the carcinogenicity
of cigarette tar with that of organic extracts of gasoline-engine
exhaust particles. The latter, obtained from a 1958 gasoline engine
without a catalytic converter, proved twice as active (on a weight
basis) as cigarette tar. Many studies have since been conducted with
skin as the target tissue; only a few are described here.
Automobile-exhaust condensate has been partitioned into a number of
fractions by Pott et al.,135 with the PAHs predominantly found in
fraction IV, the nitromethane phase. Each of these fractions was
testedl5 for ability to produce papillomas and carcinomas in life-
long mouse skin-painting experiments in which combined initiator and
promoter activity was measured. BaP, the positive control, at
1.92-7.68 ~g/treatment caused tumor formation in 15-60% of the mice.
The exhaust condensate at 0.53-4.7 mg/treatment, equivalent to BaP at
0.15-1.35 ~g/treatment, produced tumors in 1-72% of the mice, and the
tumors arose after a shorter latent period. The major tumor-producing
activity was noted in fraction IV, which contained the PAHs. In this
fraction, however, BaP is responsible for only 91 of the carcino-
genicity of automobile-exhaust condensate (AEC). 4 Agents other than
BaP, acting either alone or synergistically with AEC, are responsible
for the major carcinogenicity of AEC and probably of diesel exhaust.
The tumor-producing effects of AEC in the carcinogen mouse model
have been contrasted with those of 15 PAHs that occur as major
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components of AEC.~14 These components and their relative
concentrations in a simulated AEC mixture are shown in Table 4-18.
AEC, diesel-exhaust condensate (DEC), BaP (the positive control), and
the mixture of PAHs were tested for their comparative potency (see
Table 4-19~. The data indicate the greater potency of AEC than of
DEC. If the relative potency of AEC were accepted as 1, the
corresponding values for DEC, BaP, and the PAR mixture would be 0.02,
187, and 68, respectively. The proportions of the carcinogenic potency
of AEC and DEC attributable to the selected PAHs can be calculated.
BaP would account for only 9.6% of this potency in AEC, and the
selected PAHs, only 41%. In DEC, the contribution of BaP is
approximately 161. These results indicate that compounds other than
the selected PAHs contribute to the carcinogenic potency of AEC or DEC.
Slaga and associatesl58 used a mouse that had been bred for
quickness of response in the initiation-promotion skin-carcinogenesis
model--the SENCAR mouse--to study comparative biologic potency of
various kinds of emission and PAHs (see Table 4-20~. The exhausts were
relatively ineffective, in comparison with purified BaP, in causing
papilloma formation. Indeed, 10 mg each of emission from roofing tar,
coke ovens, and the Nissan diesel engine was equivalent in response to
50, 60, and 80 g of BaP, respectively. In no case did 10 mg of
emission extract contain that much BaP. The activity of anthracene,
pyrene, dibenz~ah~anthracene, dibenz~acianthracene, benz~aJanthracene,
2-hydroxybenzota~pyrene, and BaP as complete carcinogens and as tumor
initiators was compared in this mouse strain. 158 Their relative
potencies were 0, 0, 20, 0, 5, 30, and 30, respectively, compared with
7,12-DMBA, set at a potency of 100. Schmahl and colleagues extended
these studies by determining whether groups of nonactive PAHs would
interact with the carcinogens in a synergistic or inhibitory
manner.149 The proportions of the various compounds were chosen on
the basis of their relative concentrations in automobile exhaust. The
groups of carcinogens and noncarcinogens are shown in Table 4-21, and
the percent tumor formation after lifetime application is shown in
Table 4-22. Mixtures of the four carcinogens were more effective than
a comparable dose of BaP alone. Of greater importance, no evidence of
synergism or inhibition could be found when mixtures of carcinogens and
noncarcinogens were applied.
The application of multiple PAHs to mouse skin has often resulted
in data that were confusing, with regard to carcinogenesis. Thus, in
opposition to the above discussion, Steinerl60 reported that the
combination of two weak carcinogens, benz~ajanthracene and chrysene,
resulted in a synergistic-tumorigenic response; benz~aJanthracene and
dibenz~ahianthracene yielded fewer tumors than expected; and
dibenz~ahianthracene and 3-MC yielded the sum of individual tumorigenic
potentials. Falk and co-workers47 reported much lower tumor
production after the simultaneous administration of BaP and3severa
noncarcinogenic hydrocarbons. Van Duuren and Goldschmidt17 noted
that repeated application of the weak carcinogen BeP and the
noncarcinogen pyrene to mouse skin with BY resulted in a
cocarcinogenic effect. DiGiovanni et al. 1 found that mouse-skin
carcinogenesis induced by 7,12-DMBA-~is inhibited when BeP, pyrene, or
fluoranthene was applied 5 min before the initiator. The apparent
paradox was explained by the later studies of DiGiovanni and
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Slaga.40 They used either 7,12-DMBA, BaP, or 3-MC as an initiator
and tetradecanoyl phorbol acetate (TPA) as the promoter. BeP or
dibenz~ac~anthracene was applied 5 min before the initiator in all
cases. With 7,12-DMBA as the initiator, BeP and dibenz~acianthracene
each reduced tumorigenes is by more than 80%. However, with BaP as
. . .
initiator, dibenz~ac~anthracene exerted no effect and BeP stimulated
tumor formation by 30%. If dibenz~ac~anthracene was applied 12, 24, or
36 h before BaP, a reduction in tumorigenesis was observed. With 3-MC
as initiator, dibenz~acianthracene inhibited tumor formation, whereas
BeP was without effect. BeP apparently exerts its effect on the
7,12-DMBA-initiated system by profoundly inhibiting the ring
hydroxylation of this initiator and reducing the covalent binding to
DNA. Thus, the order of application of the multiple noncarcinogenic
with carcinogenic PAHs can have serious effects on carcinogenesis.
Finally, with regard to mouse-skin tumorigenesis, cyclopentatcd]-
pyrene, a major component of soot that can transform mouse fibroblasts
oncogenically,122 was tested for tumor-initiating activity on mouse
skin by Wood et al. 181 Although tumorigenic, cyclopentatcdipyrene
was weaker than BeP.
TISSUES OTHER THAN SKIN
. . .
PAHs and exhaust condensates have been administered to experimental
animals in ways other than topically. The subcutaneous injection of
AEC and fractions thereof into mice produced sarcomatous lesions;135
administration of 20-60 mg yielded tumors in up to at of mice, and
administration of 10 or 90 fig of BaP yielded tumors in 17t or 75t of
the animals, respectively. Simultaneous administration of 20 mg of AEC
with 90 fig of BaP yielded lower tumorigenesis. The most active
fraction from AEC was the nitromethane phase, which contained the
various PAHs.
Sellakumar and Shubikl5l studied benz~aJanthracene, benzo~b]-
fluoranthene, dibenz~ah~anthracene, dibenzotai~pyrene, and pyrene.
They mixed the PAHs with a hematite dust (at 1:1), suspended the
mixture in 0.9% saline, and instilled it intratracheally at weekly
intervals into Syrian golden hamsters. Most of the PAHs were not
carcinogenic in this limited series , but dibenzotai~pyrene produced a
high incidence of carcinomas. With multiple doses that totaled 8 mg of
this subs Lance, 47: of the hams ters had respiratory tract tumors
(squamous cell carcinomas); with 12 ma, 89% of the animals were
affected. This degree of carcinogenicity is greater than that of BaP.
Reznik-Schuller and Mohrl37 have compared the carcinogenicity of
AEC with that of several major PAR constituents in the Syrian golden
hamster intratracheal model. The hamsters were given AEC at 2.5 or 5
mg/animal every 2 wk intratracheally, corresponding to a total
administration of 42.5-75 or 75-150 mg of AEC. The total was
equivalent to 11.56-25.5 or 25.5-51 fig of BaP. In all animals,
multiple pulmonary adenomas were observed. This strikingly high
incidence of neoplasia could not be explained by the BaP content of the
AEC, It is of interest, however, that no carcinomas were observed.
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As indicated earlier and as is discussed more fully in Chapter 6,
ingestion of PAHs, whose presence may be attributed to vehicular
exhaust, appears to be a ma jor route of entry in animal systems. Yet,
the literature pertinent to this form of administration of exhaust
particles, their major PAHs, and mixtures thereof is very limited.
Neal and Rigdonl2l, 40 have examined the effects of oral administra-
tion of BaP on tumor formation in mice. No gastric tumors developed in
any of the 289 mice that were fed a control ration; the incidence of
tumors in the BaP-fed mice depended on concentration in the food and on
the number of days of feeding.l2l These investigatorsl40 also
established that the incidences of pulmonary adenomas, gastric tumors,
and leukemia in BaP-fed mice were genetically determined. No relation-
ship, however, was observed between the relative incidences of these
two types of neoplasms within a given mouse. Studies of these types
would be useful, with regard to other PAHs and their mixtures. The
interpretation of these studies is colored by the failure to house the
mice in metabolic chambers, which would eliminate the contribution of
coprophagy.
Another series of studies took advantage of the susceptibility of
the A strain mouse to pulmonary adenoma formations particularly after
the intravenous administration of selected PAHs.1 3 Shimkin and
Stonerl53 were able to calculate the amount of each agent that had to
be injected for the induction of one pulmonary adenoma in this strain
of mouse. The compounds tested were 3-MC, dibenz~ah~anthracene,
7H-dibenzotcgicarbazoyl, BaP, dibenz~aj~aceanthrylene, and dibenz~ah]-
acridine. The respective values were 0.9, 1.O, 6.0~, 9.5, 14, and 18
mol/kg of body weight for one adenoma. Benz~aJanthracene was essen-
tially inactive. The objection to the use of the A strain mouse for
these types of studies rests on its extraordinary sensitivity to
pulmonary adenoma formation. In fact, if the A strain mouse is allowed
to survive long enough, almost all the untreated animals will develop
these tumors--they are already "initiated. "
ALKYLATED PAHs, MUTAGENESIS, AND CARCINOGENESIS
.
Because of the presence of alkylated PAHs in cigarette smoke and
various coal-derived liquids and tars,56362371373 their biologic
effects are of paramount interest. Perhaps the most thoroughly studied
of the alkylated PAHs are the methylbenz~aianthracenes,
methylchrysenes, methylanthracenes, and methylphenanthrenes.
Some of the earliest studies, in which the effects of a methyl
group on the carcinogenicity of benz~ajanthracene (BA) were
investigated, were conducted by Dunning and Curtiss and by Huggins's
laboratory.8 These investigators monitored sarcoma incidence in
rats to which-the various PAHs had been administered subcutaneously.
Their results, which were in remarkable agreement, indicated that the
insertion of a methyl group at position 6 or 7 of BA increased
tumorigenicity to the extent that 70-100t of the rats were affected.
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However, 8- or 12-methyl-BA resulted in tumor formation in only 50-69%
of the rats, and 1-, 2-, 3-, 4-, 5-, 9-, 10-, or 11-methyl-BA proved
noncarclnogenlc.
The nature of the alkyl group was an important consideration:
substitution of an ethyl group at position 7 or 12 of BA greatly
diminished tumor incidence, compared with that of the methyl
congeners.123 Pataki and Huggins 123 have also studied the
structure-activity relationship in the BA series when two methyl groups
were inserted. A marked increase in tumorigenicity--shown by sarcoma
formation--was observed with 6,7-dimethylbenz~aJanthracene (DMBA) and
6,8-, 6,12-, 7,8-, 7,12-, and 8,12-DMBA. But, 1,12-, 3,9-, and 9,10-
OMBA were essentially nontumorigenic. Of the trimethylated BA
derivatives, 6,7,8-, 6,7,12-, and 7,8,12-trimethylbenz~aJanthracenes
were all very tumorigenic.
The mutagenicity and tumor-initiating activity of methylated
fluorenes, phenanthrenes, anthracenes, and benzofluorenes
were studied by LaVoie et al.99~100 Only the 9-methylfluorene was
more mutagenic in the Salmonella/microsome assay than the parent
compounds; the 1-, 2-, 3-, and 4-methylfluorenes were as poor mutagens
as fluorene itself. In the clime thyl series, 1,9-dimethylfluorene was a
potent mutagen in Salmonella TA 100, and the 2,3- and
9,9-dimethylfluorenes were relatively ineffective. Benzo~a~fluorene,
benzotb~fluorene, and benzo~cifluorene were poor mutagens in the
organism, but the 11-methyl derivatives of the first two and the
7-methyl derivative of the latter were more effective. Of this series
of methyl derivatives, 11-methylbenzo~bifluorene was the best mutagen.
In the phenanthrene series, 99 only the 1- and 9-methyl analogues
exhibited greater mutagenicity than phenanthrene itself. Equal
mutagenic activity was manifested by phenanthrene, the 2-, 3-, and
4-methyl analogues, and the 3, 6- and 2, 7-dimethyl analogues . The poor
mutagenic activity of anthracene was not altered by substitution of a
methyl group in position 1, 2, or 9.
Tumor-initiating activity of several of these alkylated PAHs was
determined with the mouse-skin two-stage carcinogenesis model.l°° In
a series of fluorene, 9-methylfluorene, 1,9-dimethylfluorene,
benzota~fluorene, benzo~b~fluorene, benzotcifluorene,
11-methylbenzo~a~fluorene, 11-methylbenzotbifluorene, and
7-methylbenzotcifluorene, only 11-methylbenzotb~fluorene resulted in a
marked increase in tumorigenicity. All other compounds exhibited
rather weak initiator activity.
The methylchrysenes are known respiratory pollutants that occur in
substantial amounts in cigarette smoke--approximately 18
ng/cigarette.62 Although chrysene itself is generally inactive,
several of the meghylated species are carcinogenic. In early studies,
Gough and Shoppee 5 and Dunlap and Warren44 showed that the 1-, 4-,
4-20
OCR for page 155
and 6-methylchrysenes demonstrated only weak tumorigenicity. The
1,11-dimeth~1 derivative, however, was moderately active as a skin
carcinogen, 2 although less so than 3-MC.
Hecht and colleagues63 studied a series of methylated chrysenes
as both complete carcinogens and initiators. As a complete carcinogen,
5-methylchrysene was far superior to chrysene and the other
monomethylated derivatives; it was almost equivalent in carcinogenic
potency to BaP. 2-Methylchrysene exhibited about 50% of the carcino-
genicity of the 5-methyl analogue . As an initiator, 5-methylchrysene
was also the most potent of the methylated derivatives, yielding tumors
in 50% of the mice by 14 wk. Next in potency was 3-methylchrysene.
The 1-, 4-, and 6-methylchrysenes were all much less effective as tumor
initiators. These investigators63 considered whether 5-methyl-
chrysene, rather than BaP, would be a major contributor to the carcino-
genicity of tobacco smoke; but, in view of its small concentration in
tobacco smoke, compared with that of BaP (0.6 ng/cigarette vs. 30
ng/cigarette), it is unlikely that this is so.
A series of methylated BaPs were tested for tumor-initiating
activity with the mouse-skin carcinogenesis model.157 Several of the
methylated derivatives exhibited greater initiating activity than the
parent compound, namely, the 1-, 3-, and 11-methyl analogues. Several
were completely ineffective in this regard: the 7-, 8-, 9-, -and
10-methyl analogues. The 4-methyl derivat ive was about equal to BaP in
initiating potency.
From these examples, it is apparent that some methylated PAHs are
strong carcinogens and therefore should be reckoned with as environ-
mental contaminants.
TOBACC O- SMOKE CARC INOGENE S I S
.
Although the topic has been discussed extensively, several of the
potent carcinogenic PAHs that are present in tobacco smoke should be
mentioned here .
Approximately half of tobacco smoke consists of particulate consti-
tuents in which over 2,000 compounds are represented. The carcino-
genicity of cigarette smoke was demonstrated ~hrough skin application
to the backs of mice and the ears of rabbits7 and has been confirmed
repeatedly in a number of laboratories. Unfortunately, inhalation
experiments have not led to as clear-cut a conclusion. When Syrian
hamsters were exposed to diluted smoke (smoke-to-air ratio, 1:7) for 10
min twice a day for 18 mo, precancerous lesions were observed in 30%,
esophageal tumors in 5t, and laryngeal carcinomas in 10% of the
animals; no bronchial or tracheal cancer was seen.72
Cigarette-smoke condensates have been partitioned into a number of
fractions, of which the most carcinogenic is the "neutral fraction,"
representing 57: of the mass of the condensate.72 Although the
OCR for page 196
175. Wallcave, L., D. L. Nagel, J. W. Smith, and R. D. Waniska. Two
pyrene derivatives of widespread environmental distribution:
Cyclopentatcd~pyrene and acepyrene. Environ. Sci. Technol. 9:
143-145, 1975.
176. Wailer, R. E. The benzpyrene content of town air. Brit.
6:8-21, 1952.
177. Wang, Y. Y., S. M. Rappaport, R. F. Sawyer, R. E. Talcott, and
E. T. Wei. Direct-acting mutagens in automobile exhaust. Cancer
Lett. 5:39-47, 1978.
178. Whong, W-Z., J. Stewart, and T-M. Ong. Use of the improved
arabinose-resistant assay system of Salmonella typhimurium for
mutagenesis testing. Environ. Mutagen. 3:95-99, 1981.
179. Williams, G. M. The detection of chemical mutagens/carcinogens
by ONA repair and mutagenesis in liver cultures, pp. 61-79. In A.
Hollaender and F. DeSerres, Eds. Chemical Mutagens: Principles and
Methods for Their Detection. Vol. 6. New York: Plenum Press, 1980.
180. Wolff, S. Sister chromatic exchange. Ann. Rev. Gene t. 11:
183-201, 1977.
181. Wood, A. W., W. Levin, R. L. Chang, M-T. Huang$ D. E. Ryan,
P. E. Thomas, R. E. Lehr, S. Kumar, M. Koreeda, H. Akagi,
Y. Ittah, P. Dansette, H. Yagi, D. M. Jerina, and A. H. Conney.
Mutagenicity and tumor-initiating activity of cyclopenta~c,d)-
pyrene and structurally related compounds. Cancer Res. 40:
642-649, 1980.
182. Wynder, E. L., and D. Hoffmann. A study of air pollution carcino-
genesis. III. Carcinogenic activity of gasoline engine exhaust
condensaste. Cancer 15: 103-108, 1962.
1830 Wyrobek, A. J., and W. R. Bruce. Chemical induction of spenm
abnormalities in mice. Proc. Natl. Acad. Sci. USA 72:4425-4429, 1975.
184. Yasuhira, K. Damage to the thymus and other lymphoid tissues
from 3-methylcholanthrene, and subsequent thymoma production, in
mice. Cancer Res. 24: 558-569, 1964.
J. Cancer
4-62
~ . .. .. ..
OCR for page 197
5
EFFECTIVE BIOLOGIC DOSE
In the class of polycyclic aromatic hydrocarbons (PAHs), there are
several chemicals that are environmental pollutants; some are carcino-
genic in experimental animals, and some are suggested to be carcino-
genic in humans.160 In the body, they are enzymatically converted to
reactive forms that bind extensively and covalently to cellular macro-
molecules.63~80~15l~l82 The covalent binding of reactive metabolites
of PAHs to DNA is considered to be an essential first step in PAH
induction of neoplasia.63~80~84~128~151~182 The damaged DNA cannot
be fixed and results in a mutation within the cell unless enzymatic
repair occurs first. There are many phanmacokinetic and enzymatic
processes involved before the formation of reactive metabolites of
PAHs, which may ultimately form adducts with DNA. Thus, the
concentration to which a person is exposed is probably not a good
measure of the biologic dose that causes neoplasia7~6 ,149 or other
PAH-induced toxicoses (see Chapter 4~. This chapter develops the theme
that some degree of PAH metabolite-DNA adduct formation in the target
tissue can be used as a measure of effective biologic dose. The
effective biologic dose of a substance is a reflection of its
absorption, distribution, metabolism (activation or detoxification),
and excretion. In the case of an alkylating substance, such as a PAH,
that dose can be measured directly on the basis of the amount of
alkylated DNA, itself a reflection of adduct formation. If the
accumulation of adducts in DNA is greater than the capacity of the
tissue to repair such lesions accurately and greater than the capacity
of the tissue to replicate its DNA, then the presence of adducts will
be indicative of the effective biologic dose. The chapter begins with
a brief discussion of the pharmacokinetics of PAHs. That is followed
by a discussion of the metabolism of selected PAHs. The in viva
formation and disappearance of PAH metabolite-DNA adducts are next
reviewed in detail. Finally, there is a discussion of the possibility
of using PAH metabolite-DNA adduct content as a measure of effective
biologic dose for in vitro mutagenesis, initiation of carcinogenesis,
and inhibition of replication and transcription.
PHARMACOKINETICS
Many phanmacokinetic and enzymatic processes are involved before a
PAH reaches a target cell and is metabolized to reactive metabolites
that interact with DNA and other cellular macromolecules free Figure
5-1~. The oxidative metabolism of PAHs is usually by cytochrome P-450,
and the formation of excretable glutathione, glucuronide, and sulfate
conjugates results in a very complex metabolic profile. Thus, pharma-
cokinetic information that would enable one to construct mathematical
5-1
OCR for page 198
modern of the tissue distribution, metabolism, covalent binding to
cel lular macromolecules , and excretion of PAHs and metabolites as func-
tions of exposure dose are nonexistent. However, sufficient studies
have been done to allow some general ization regarding absorption, tis-
sue distribution, and elimination of PAHs (see Santodonato et al.,160
pp. 6-1 through 6-27~. Most of these studies have only followed radio-
activity in various tissues, urine, and feces after administration of
radiolabeled PAHs.
PAHs are readily absorbed after administration by various routes
and are then rapidly removed from the blood and distributed into a
variety of boa: tissues. Kotin et al.l09 examined the radioactivity
derived from I C-labeled benzota~pyrene (BaP) in various tissues of
rats and mice after intravenous, subcutaneous, and intratracheal
administration. The blood concentrations resulting from intravenous
injection were hardly detectable after 10 min. Radioactivity was found
in stomach, intestine, liver, kidney, lung, spleen, testis, myocardium,
urine, and feces. The pattern of distribution was independent of route
of administration, except that particularly high lung concentrations
followed intratracheal administration. These workers did not examine
fat or mammary gland. Other investigators have shown that nonmetabo-
lized BaP,19 3-methylcholanthrene (3-MC),19~40 and dimethylbenz~a]-
anthracene (DMBAJ19~58 accumulate and persist more in fat and mammary
tissue than in other tissues. Some PAHs induce neoplasia in the
mammary glands of rats.
Rees et al.l57 examined the mechanisms by which BaP and other
PAHs are absorbed from the gut. Accumulation of BaP in averted sacs of
small intestine increased exponentially with incubation-medium
concentration. The transport of BaP from the sac tissue to the inside
medium was found to be proportional to the concentration in the sac
tissue. Thus, if the capacity of other tissues to absorb BaP from
extracellular fluid (and blood) is proportional to the concentration of
BaP in the fluid, then accumulation in the tissues should also be
proportional to intragastric concentration. For example, this
relationship was observed in adipose and mammary tissue 18 h after oral
administration of BaP. Rees et al. postulated-a mechanism of physical
_ _
adsorption onto the intestinal mucosal surface and then passive
diffusion into and through the intestinal wall. The proportional
nature of the accumulation in the t issue can be accounted for by two
phases of adsorption, one unilayer and the other multilayer. Even if
tissue accumulation of PAHs is proportionally related to exposure dose,
these results should not be overinterpreted. The situation is dynamic;
the accumulation is transient, in that PAHs are rapidly metabolized and
removed from the body. Rees _ al. observed that BaP disappeared very
rapidly from the thoracic duct lymph. Moreover, PAR metabolite-DNA
adduct content in various tissues is not linearly related to exposure
dose (as discussed later).
A relevant route of environmental exposure to PAHs is deposition in
the lung of particles with PAHs on their surfaces. In general, the
degree of resent ion of PAHs in the lung is a function of the size and
5-2
OCR for page 199
composition of the particles carrying them. Several investigators have
shown that BaP retention by the lung is higher when it is adsorbed on
particulate carbon 39~85 dust,l64 ferric oxide,85 aluminum
oxide, 85 and talcl45 than when it is not; carbon-particle size
affects BaP retention, but the size of particulate ferric oxide or
aluminum oxide does not. 5 However, some recent studies have sug-
gested that particulate adsorption of PAHs does not alter retention
time in the lung or their distribution to other tissues. Adsorption on
ferric oxide did not increase the retention time of BaP in hamster lung
after intratracheal instillation.57 Pylev et al.l55 examined the
clearance of intratracheally instilled BaP from the hamster lung; the
disposition and clearance from liver, kidney, and blood; and excretion
into feces and urine. BaP was instilled alone or adsorbed on asbestos
or carbon black. Although these studies were limited in scope, it was
found that the disposition of BaP from lung to other tissues, the rate
of tissue clearance of BaP, and the pattern of BaP excretion were not
altered by the introduction of BaP into the hamster either in free form
or bound to particles. Obviously, more studies on rates of clearance
from the lung and the later fate of particle-adsorbed PAHs are needed
to clarify the effects of particle size and composition. However, it
can be concluded that distribution to other tissues occurs after
pulmonary exposure to particles on which PAHs are adsorbed.
Elimination of PAHs in animals occurs mainly by excretion of con-
jugated metabolites into the feCeS.4,23,109,161,162 There is so
excretion of metabolites into the urine--approximately 10% in the study
by Kotin et al.l09 Excretion into bile can be very rapid. For
example, 6 h after intravenous injection of [3H]BaP, 60-70t of the
tritium appeared in bile or conjugated metabolites.23 PAR clearance
from an animal probably is not limited by metabolic rates or biliary
clearance of metabolites, but rather is affected by the persistence of
nonmetabolized compound in various tissues (such as fat, skin, and
mammary gland) or perhaps by adsorption on particles.
The pharmacokinetics of a PAH will be influenced by prior treatment
with chemicals capable of inducing enzyme systems that metabolize it.
Schlede et al.l61~162 have shown that pretreatment of rats with
unlabeled BaP markedly increased the plasma-disappearance rate of a
tritiated dose of the same compound given intravenously; the effect was
especially marked during the first 5 min after the intravenous
administration of the radiolabeled material, and increased clearance
lasted for 6 h. This effect of pretreatment with the compound was
paralleled by a lower concentration of [3H]BaP in brain, liver, and
fatty tissues; similar but more variable results were observed in lung
tissue. These influences of BaP pretreatment on a later intravenous
dose of t3H]BaP were also observed when the radiolabeled compound was
administered orally. 3-MC and DMBA pretreatment of animals produced
comparable effects on the metabolic disposition and tissue content of
radiolabeled BaP. Pyrene and anthracene pretreatment had little or no
such effect on the in viva disposition of this compound, nor (id
phenobarbital. In other studies, the biliary excretion Of [1 C]BaP
OCR for page 200
was shown to be increased by pretreatment with the unlabeled compound;
however, no increase in excretion of the 14C-labeled metabolites of
BaP into bile was observed after pretreatment with this compound.
These findings suggest that conversion of BaP to its metabolites may be
the rate-limiting step in its biliary excretion.
METABOL I SM OF PAH s
. .
An organism's processing of xenobiotic chemicals is determined by
their physical and chemical characteristics. Figure 5-2 summarizes the
possible events leading to carcinogenesis in a cell exposed to a
xenobiotic toxic chemical. After uptake, the cell may simply excrete
the chemical unchanged, as is the case with some metals and apparently
inert materials, such as asbestos. A toxicant may contain functional
attachment groups, such as hydroxyl or ketone, that can be conjugated
to deactivating moieties like glutathione or glucuronic acid by
cytoplasmic transferase. If the toxicant is a PAH or other relatively
stable molecule, it will be attacked by the microsomal monooxygenases
and form an electrophilic intermediate, which can later be conjugated
to a deactivating moiety, detoxified, and excreted.
Once an activated electrophile is formed, it can readily attack
nucleophilic sites other than the detoxifying substrates, such as
nucleic acids and proteins. The formation of adducts between
electrophile metabolites of PAHs and UNA is probably a necessary first
step in the initiation of carcinogenesis by PAHs. The in vivo
formation of PAH metabolite-DNA adducts is discussed later in this
chapter.
These biochemical changes to biologically active intermediates
depend on the balance between enzyme systems: those enzymes generating
and those detoxifying the intermediates. One of the major enzymes
involved in activation is aryl hydrocarbon hydroxylase (AHH). It is
found in virtually all eukaryotes (and some prokaryotes), has a wide
range of specificities for substrate activity, uses a variety of
iron-containing cytosolic pigments as the active sites for chemical
oxida&~on (e.g., cytochrome P-450), and is substrate-induc-
ible. ,134 Many PAHs are capable of inducing one or more forms of
cytochrome P-450. There is some evidence that induction is regulated
by one ~ene ~: a relatively small number of genes in animal-model
systems 10,1 and perhaps even in humans (see Chapter 7~. The basis
for genetic regulation appears to reside in a balance of inducers and
receptors that are activated by PAH metabolites; after binding, trans-
location to the nucleus, expression of induction-specific RNA, and
protein s~nthesis, the generation of specific cytochrome P-450 is
observed. In the murine-model systems, genetically controlled AHH
activity is correlated with cancer formation caused by PAHs, such as
BaP,1lO 3-MC,1l0 dibenz~aJanthracene~lll and DMBA.1l0
5-4
OCR for page 201
Examples of enzymes that can detoxify these metabolic intermediates
are UDP-glucuronosyltransferase, glutathione-S-epoxide transferase,
aryl sulfatase, and epoxide hydrase. These enzymes catalyze the
conjugation of the primary oxidative species formed as a result of AHH
activity to forms that are sufficiently polar to be excreted from cells
and from the body. Some of the conjugating enzymes are also under a
form of genetic control, 142 but their role in PAH carcinogenesis is
not completely defined. Epoxide hydrase is one of the enzymes that had
-been thought to function in a manner that results in the detoxification
of PAHs; however, it is now established that, for a variety of PAHs,
epoxide hydrase can catalyze the formation of dihydrodiol derivatives
of PAHs and that these dials may serve as substrates for monooxygenase
activity again--resulting in the formation of diol-epoxides.63 The
diol-epoxides constitute at least one of the ultimate mutagenic and
carcinogenic forms of PAHs.
Over the last decade, BaP has been the most'exhaustively studied
PAH carcinogen and has been the prototype compound in developing the
mechanism of action of the cellular monooxygenase and cytoplasmic
transferases necessary to activate and detoxify PAH carcinogens. A
recent exhaustive summary of BaP metabolism dealt with its activation,
carcinogenesis, and role in the regulation of mixed-function oxidases
and related enzymes.63 A composite of metabolic products of BaP is
shown in Figure 5-3. BaP has been studied in a large number of in viva
and in vitro systems, as well as in cell-free preparations using
homogenates, microsomal fractions, and purified enzymes. BaP may form
epoxides at several sites around its ring system, and three epoxides
(4,5-,7,8-, and 9,10-) have been identified. Research over the last
half-decade has implicated the 7,8-diol (bay region*~94 as the
primary precursor for the second round of activation by mixed-function
oxidases, both cytoplasmic and nuclear,80 that form the highly
electrophilic 7,8-diol-9,10-epoxide (Figure 5-4), which opens to form a
trial carbonium intermediate. This reactive molecule has been shown to
be the major species that binds to nucleic acids via the C-10 position
of BaP and to exocyclic amino groups of guanine.
Metabolism of many PAHs other than BaP has also been shown to
proceed via diol-epoxides, such as benz~a]anthracene, l74'l88
chrysene~ll6,l89 dibenz[ah]anthracene,1 0 5-methylchrysene 81
7 ~ethylbenzanthracene (7-MBA)~35~127 DMBA,16~46$91~132~174 and
3~MCe 105, 179 The ease of formation of carbonium ions by these
diol-epoxides parallels the observed biologic activity of the parent
chemic'al~.115 Metabolic profiles on some PAHs other than BaP are
available, and salient features of their metabolism are presented below.
*The bay region is a molecular region between adjacent fused aromatic
rings (see reference 115~.
5-5
OCR for page 202
BENZO[e]PYRENE
Benzo~e~pyrene is a marginally carcinogenic structural isomer of
the strong environmental carcinogen BaP. It contains two bay regions
and, by theoretical calculations, should approximate BaP in carcino-
genic activity. Metabolic studies have determined that the prob-
able reason for its lack of carcinogenicity is that its major metabo-
lism is distal to the bay region, so that the molecule does not favor
formation of diol-epoxide intermediates. Its metabolism has been
studied in hamster embryonic eel is and in eel 1-free preparations from
rat liver. Its ma jar metabolize is 4,5-dihydro-4,5-dihydroxybenzo-
[e ~ pyrene. Large-ecale experiments with microsomes positively identi-
fied 9~10-dihydro-9,10-tihydroxybenzo~e] pyrene , but i t constituted le 8 8
than 1: of the total metabolites .
~1
"nzo 1 e ~ p~rrene
ARC
H
. ~
5-6
HO_~
H At
loJ
4,5-Dlhydro-4,5-
t thydroxy
~X?°H
9, lO~bydroxy
OCR for page 203
PYRENE
Early studies on pyrene metabolism were in rats and showed
increased urinary excretion of sulfuric acid esters and glucuronic acid
conjugates. Later, 1-hydroxypyrene and 1 )6-dihydroxypyrene were
identified.78 More definitive studies of pyrene metabolism were
performed in rabbits and rats by analysis of urinary metabolites after
intraperitoneal in Section. 172 No direct structural analysis was
performed , but the resul ts o f a number o f chromatographic and spectral
analyses were compared with synthetic s tandards. 1-Hydroxypyrene, 1,6-
and 1,8-dibydroxypyrene, 4 ,5-dihydro-4 ,5-dihydroxypyrene , and N-acetyl-
S-~4,5-dihydro-4-hydroxy-5 -pyrene ~ -L-c y s te ine were identified. The
latter compound was also isolated from bile in rats. More recent
studies with gas-liquid chromatography and mass spectrometry have con-
firmed the presence of 1-phenolic and 1-dihydroxydihydro derivatives
from rat-1 iver microsomal incubation and shown a marked increase in
mutagenesis in the Salmonella T 90 and T 100 strains.79
'~3
:0H
HO H
4, 5-Dihydro
4, 5 - ihydroxypyrene
~1
Colon
~OH
N -acetylcystelne
RE N7. f ~ 1 A NTHRACENE
Pyrene ~
~OH
H O ~
1, 8-D1 hydroxypyrene
Benz[a]anthracene is a marginally carcinogenic PAN that has both
bay-region and K-region areas. The original metabolic studies with
benz~a~anthracene were done with thin-layer
chromatography .21 ,26-28 ,75, 169, 171 (See reference 115 for explana~
Lion of the K-region. ~
5-7
~ ˘0H
[me'
O
H O ~
I, 6-D1 hyd r oxypyrene
OCR for page 204
OH
3-tlydroxy
1
OHM;
S-Bydroxy
HO
H on
8, 9-Dlhytso-8, 9~dltydroxy
Benz 1e ]anthracene
.;
S. 6-Dthy dro-5, 6 , ~dlhydroxy
These ~ tudies unequivoca 1 ly ident if fed 5, 6-dihydro-5, 6-d ihydroxybenz ~ a ~ -
anthracene and 8,9-dihydro-8,9-dihydroxybenz~aJanthracene as major
metabolites. Also reported were 1,2-dihydrodihydroxybenz~aJanthracene,
3-hydroxybenz~aJanthracene, 4-hydroxybenz~aJanthracene, and benz~a]-
anthracene 7,12-quinone. In viva ~tudiesl9l with rats, rabbits, and
mice reported a mercapturic acid derivative, presumably as a breakdown
product of a cysteine conjugate. Also reported were trace amounts of
5-8
HO ~
SHOW
A:
_~-
1, 2-Dlhydro-1, 2-
dthydroxy
~OH
OH
3, 4-Blhydro-3, 4-
dthydrosy
OCR for page 205
sulfate and glucuronic conjugates at the 3, 4, 8, and 9 positions,
presumably as products of phenols; and 10,11-dihydro-10,11-dihydroxy-
benz~ajanthracene were also reported. The 3,4-dihydro-3,4-dihydroxy
derivative of benz~aJanthracene was later confirmed with high-pressure
liquid Chromatography as a metabolite formed by rat-liver micro-
somes.1 7 This 3,4-dihydrodiol adjacent to a bay region leads to the
idea of benz~aJanthracene bay-region activation, including the possi-
bility of an isolated double bond in the 1,2 position after formation
of a diol-epoxide. That 3,4-dihydro-3,4-dihyroxybenz~aJanthracene is a
minor product quantitatively, as opposed to the less active 5,6-diol,
may explain the weak carcinogenicity of benz~aJanthracene.
CHRYSENE ( 1, 2-BENZOPHENANTHRENE )
.
This molecule is composed of two linearly annellated rings formed
by pyrocondensation of carbonaceous malarial and is therefore present
in coal tar in substantial quantities. Metabolism of chrysene has
been studied in rodents and in cell-free and organ-culture systems.
Incubation with rat-liver microsomes produced a series of hydroxylated
OH
Chrysene
Hi;
1,2-Dihydro
OH
> ~5, 6-Dihydro
~OH
3, ~Dihydro
5-9
OCR for page 206
metabolizes, as seen by high-pressure liquid-chromatographic (HPLC)
separation. Several of these metabolites have been identified with the
use of synthetic standards.122 Three dihydrodiols have been char-
acterized: the 1,2-, 3,4-, and 5,6-dihydrodiols. This metabolic
profile has been concerned with the use of rat or mouse skin-organ
culture.l23 The dihydrodiol metabolites are presumably formed
through reactive epoxide intermediates by the P-450 mixed-function
oxidases. However, no phenolic or quinoid structures have been
identified from the remaining peaks in the HPLC separation.139
5-ME:THYLCHRYSENE
Of all the methylchrysenes studied, only 5-methylchrysene shows any
substantial carcinogenicity. Metabolism of this compound has been
studied in the 9,000-g supernatant from rat liver.81~83 Liver homo-
genates used for this work were prepared from Aroclor-treated male
F-344 rats, and HPLC of metabolites showed nine peaks, of which seven
had been identified (according to their relative abundance) as
5-hydroxy-5-methylchrysene, 5-methylchrysene 1,2-diol, 7-hydroxy-5-
methylchrysene, 5-methylchrysene 9,10-diol, 9-hydroxy-5-methylchrysene,
1-hydroxy-5-methylchrysene, and 5-methylchrysene 7,8-diol. Two minor
metabolites have not been identified. The bay-region theory would
predict that 5-methylchrysene 1,2-diol and 5-methylchrysene 7,8-diol
are primary candidates for active carcinogenic intermediates. However,
experiments with liver homogenates indicated that formation of 5-methyl-
chrysene 1,2-diol is favored over that of 5-methylchrysene 7,8-diol.
No other biologic system has been used to study metabolism of 5-methyl-
chrysene, so it is not possible to make any pertinent comparisons with
other tissues or between intact-cell activation and detoxification.
5-10
~. .... .
Representative terms from entire chapter:
sister chromatic
