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OCR for page 207
OH
0R
.
OH
OH
\ ~1, 2-Dlhydroxy
5-Me thylchrysene
HO H3
OUCH ~7,8-Dihydro
5-Bydroxy-H >~
9''10-Dlhydro-9, 10-tihydroxy
FLUORENONE
There appear to have been no direct studies on the metabolism of
fluorenone~ but N-3-fluorenyl acetamide (3-FAA) yielded two metabolic
products.] 2 Because the parent compound is carcinogenic, it appears
that the derivatives are detoxification products. The authors sug-
gested that metabolism of 3-FAA consists of two sequential reactions:
the initial formation of 9-hydroxy-3-F M as an intermediate to the for-
mation of 3-acetamido-9-fluorene hydroperoxide, which is then dehydrog-
genated to form 9-oxo-3-FAA. Exposure of rainbow trout to a number of
hydrocarbons showed no bioaccumulation of fluorenone; the compound is
most likely metabolized to excretible products.65 However, no at-
tempt was made to analyze any metabolic products. There is no litera-
ture on the isolation and identification of fluorenone metabolites.
S-11
7, 8-dihydroxy
OCR for page 208
-
- o
9-Fluorenone
o
l o ~ N-C-cH"
H H
N-3-fluorenyl acetamide ( 3 FM)
}£E: THYLFLUORENE
(~1 ~
LO I
~0 H
1
o
3~ ~C-Cu3
9~Bydroxy-
-3 FAA
o
4~N-~-C H.
11
3-Acetamite-9-fluorene-
hydroperoxide
There is no literature on the metabolism of methylfluorene, but
there has been a major study on methylfluorene-2-acetic acid (MFA)
(Cycloprofen, Squibb Institute for Medical Research). This compound is
an anti-inflammatory agent whose metabolism has been studied in rats;
its metabolites have been isolated from urine and identified. Its
major metabolite is substituted at the 7 position on the aromatic ring,
so its metabolism may be similar to that of methylfluorene. This
congener was given both orally and intraperitoneally. Analysis of the
metabolizes by thin-layer chromatography yielded six peaks, of which
four have been identified. The ma jar metabolite, consisting of 47% of
the material, was 7-hydroxy-MFA, with approximately 10% each of
9-hydroxy-MFA and 7,9-dihydroxy-MFA.42~112
5-12
i ... ..
OCR for page 209
CH3
rot
tie thylfluorene
-_
I O [~ O |9H3
tie thylfl uorene-
2_caticacid
HO~-C-OH
OH ~
7, 9-Dlhydroxy
CYCLOPENrA [ Cd ] PYRENE
-
-
O ~ C-tO H
7-llydroxy
~Lc i-OH
9-Nydroxy
Cyc logenta [ cd ] pyrene has been ident i f fed as a component of carbon
black.66, 9 Its metabolism has been studied in rat-liver micro-
somes.67 The major metabolize isolated by HPLC has been identified
as trane-3,4-dihydroxy-3,4-dihydrocyclopenta~ct~pyrene. Several came
portents not yet well characterized consisted presumably of phenolic
derivatives, as well as metabolites that appear to have saturation of
the ethylene bridge.
5-13
I. .. .
OCR for page 210
~-
-
~clopenta[cd]pyre~
-
DIBENZOTHIOPHENE AND BENZOTHIOPHENE
b~-
-
-
-
Dibenzothiophene and benzothiophene are biodegradable in both
eutrophic and oligotrophic pond waters.20 Their major metabolite is
1,2-dihydro-1,2-dihydroxydibenzothiophene, with later ring degradation
to benzothiophenedione. Benzothiophene and dibenzothiophene form a
thioketone, a dihydrodiol (cis and bans isomers and a diketone).
There have been no studies dealing with further metabolism.
Enzymes other than microsomal monooxygenases may also be involved
in the metabolic activation of PAHs. Eling et al.52 and Marnettl25
have shown that numerous xenobiotics, including the dihydrodiol
metabolites of PAHs, can be cooxygenated during the oxygenation of
arachidonic acid by prostaglandin synthetase. In the case of PAHs,
when the dihydrodiols are generated, this novel pathway could lead to
an alternative pathway for the formation of diol-epoxides. These
studies have been done on in vitro model systems. The relevance of
their pathway in viva is unknown.
There is a suggestion in the literature that nitropyrenes are
metabolized by bacteria, presumably via a nitroreductase, to produce a
high mutagenicity; however, there has been no isolation or character-
ization of metabolites. Because mammalian cells have much-reduced
nitroreductase activity, this rationale has been used to explain the
lack of activity in mammalian cells.62
5-14
-
Trane-3, 4-dthydroxy
'5~:
09H
3, 4-dihydrocyclopenta [ cd ] pyren.
~. .... ..
OCR for page 211
s
Dibenzothiophene
Benzo thiophene
H OH
OH
ASH
LC-H
o
-
.
a;
it'
S'
r'
o
-
5-1S
1
OH
S H
~5-0
~-so
~S)~
Benzo thiophenet lone
ILL .. -.
OCR for page 212
IN VIVO FORMATION AND DISAPPEARANCE OF PAH METABOLITE-DNA ADDUCTS
. . .
HISTORICAL PERSPECTIVE
.
Brookes and co-workers observed that 3H-labeled PAHs applied to
the backs of mice30 or incubated with mouse-embryo celis48 resulted
in covalent binding of radioactivity to DNA, RNA, and protein. Grover
and Sims76 and Gelboin62 showed that PAHs require metabolic activa
tion by mixed-function oxidases if they are to react covalently with
cellular macromolecules. The interactions between PAH metabolites and
nucleic acids have since received considerable
attentiOn.63, 80,151,182 Identification of the reactive PAH metab
olites that form adducts with DNA has been emphasized, because forma
tion of these adducts is believed to be essential for tumor initiation,
although interaction with RNA and protein may also be important.
Initial attempts to identify the reactive metabolites that bound to
DNA focused on the arene oxide intermediates proposed by Boyland,25
and especially on the K-region arene oxides, because, according to
quantum mechanics) carcinogenic PAHs are distinguished by an electron-
rich K region.l°, 53 They induce mall transformation of cells
and are active in mutagenicity tests. ~ In addition, they are
b li f pAHs70~72-74,104,163~170 and will bind to DNA in
vitro.18 However, it became obvious that K-region epoxide-DNA
adducts were not the adducts formed in viva between BaP metabolites and
DNA.18 A similar conclusion was reached in studies with 7-MBA.17
Borgen et al.22 found that, in a microsomal activating system,
the 7,8-diol BaP metabolite bound to DNA to a much greater extent than
any other known dial or phenol. Sims et al.172 provided evidence
that the BaP metabolite-DNA adduct formed by BaP metabolism by Syrian
hamster-embryo cells in culture was chromatographically ~ dentical with
an adduct formed by metabolism of BaP 7, 8-dio1 and proposed that t'ne
reactive metabo1ite was a diol-epoxide (DE). Studies in various in
vivo model systems such as cell cultures and organ
explants 13,18,29,)1,79,93,172 and in in `'ivo skin, lung. liver, and
forestomach of mice have shown that the major Ba? metabolit2-DNA adduct
observed after exposure of these tissues to BaP is the (+~-BaP DE
I-deoxyguanosine adduct. (+)-BaP DE I is apparently the major enzy-
matic rnetabolite of --trams BaP 7,8-diol. ~ The adduct results
from the interact ion of ~ + ~ -BaP DE I wi th two amino groups of guanine .
The cis isomer (-~-BaP DE II is also formed enzymaticaily from
(-~-trans-BaP 7,8-diol. 194 The --Bar DE II-deoxyguanosine adduct
is formed to the same extent as the ~ +)-BaP DE I-deoxyguanos ine adduct
in lung and liver of rabbits, as opposed to the results in mice (C.
Bixler and M. W. Anderson, unpublished data). Structures of the BaP DE
isomers are shown in Figure 5-5. Although the predominant by nding of
BaP DE is to the 2-amino group of guani8e~ these 7 iol-epoxides can also
bind to the N7 of guanine, 1 adenine, 9 , 9, 130, 1 5 and
cytidinel75 and to phosphate real dues.00 ,108
5-16
OCR for page 213
Evidence is Crating that gthg; PAHs--e.g., 7-MBAg35~127
benzanthracene,1 ~ chrYsene 1 ,1 5-methylchrysene 1
dibenzanthracene,l90 3-Mc,l05,l}9 and DMgA16,48,91,132,1~9__are
similarly converted to highly reactive diol-epoxides, which then
interact with DNA in viva. All these diol-epoxides have a structural
similarity. Jerina and Daly94 pointed out that the epoxide ring is
in a bay region and suggested the term "bay-region diol-epoxides" for
these highly reactive metabolites of PAHs.
The bay-rezion diol-enoxides are
mutagenic 89,96,115,117,126,136,174,185,186 have transforming
activity in mammalian cells,843126 are carcinogenic in newborn
mice3l~l00,l0l~ll7 and Chinese ham te 1 in cells 1173187 a d are
initiators in cells of mouse skin.~55~&0~0~,ii5~7~75 The
mutagenicity and carcinogenicity, combined with the observation that
bay-region diol-epoxide-DNA adducts are the major adducts formed in
viva in target tissue, have led to the hypothesis that bay-region
diol-epoxide adducts are the ultimate carcinogens generated by
metabolism of most PAHs.963115 However, it should be pointed out
that many nonmetabolized PAHs that are termed carcinogenic either lack
a bay-region benzene ring or contain nonreactive substitutes in this
molecular region.80 In addition, PAH metabolite-DNA adduct formation
in viva has been examined only for BaP, DMBA, and 3-MC, and these
studies have concentrated on target tissues in mice (see Table 5-1~.
PAH metabolites other than bay-region diol-epoxides can also bind
to DNA. The K-region epoxide of BaP binds DNA coval~ntly.168~170
Incubation of BaP with microsomes in the presence of exogenous UNA
results in a variety of BaP metabolite-DNA adducts.12~148 In
particular, adducts are formed from further metabolism of 9-hydroxy-
BaP, possibly the 4,5-epoxy-9-hydroxy-BaP metabolize. The
major DNA adduct observed after exposure of hepatocytes in culture to
BaP resulted from the further metabolism of 9-hydroxy-BaP.97 A
BaP-phenol-oxide-DNA adduct was the major adduct observed in rat lung
and liver after intravenous administration of BaP.23 Various
structural modifications of PAH diol-epoxide metabolites do not inhibit
binding to DNA.80,86,87
Dose-response relationships for formation of PAH metabolite-DNA
adducts in target tissue would be helpful in the low-dose extrapolation
problem for PAH carcinogenesis.7361 Many pharmacokinetic processes
determine the extent of formation of PAH metabolite-DNA adducts in an
organ after exposure of an animal to a PAH (see Figure 5-1~. Although
most of these processes have not been completely characterized, some
generalizations regarding the extent of adduct formation in viva can be
made from recent reports (see Table 5-1~. Previous reviews of covalent
binding of PAHs to DNA have not analyzed in viva adduct formation in
detail.63~80~151~182
5-17
~ . .. . ..
OCR for page 214
CHARACTERIZATION OF PAH METABOLITE-DNA ADDUCTS
. . .
Table 5-1 lists the studies concerned with the in viva formation of
PAH metabolite-DNA adducts. Most of them used mice and BaP.
HPLC analysis of BaP-deoxyribonucleoside adducts formed in lung,
liver, and forestomach of A/HeJ mice after oral administration of BaP
(3 mg/mouse) is shown in Figure 5-6.8~184 HPLC analysis is shown for
DNA samples isolated by the hydroxylapatite and precipitation pro-
cedures. Radioactivity elated in the water wash (water fraction, WF)
and in early portions of the water:methanol gradient that varied from
407 to 70%. This uncharacterized early-etuting radioactivity was much
higher in UNA samples isolated by precipitation than by the hydroxyl-
apatite method (see Figure 5-6), although it was still substantial,
especially in liver and forestomach, in samples isolated by the
hydroxylapatite procedure. Three distinct peaks--I, II, and III in
Figure 5-6--were observed id the gradient portion of the chroma-
tography. The specific activity (picomoles per milligram of DNA) asso-
ciat~d with these peaks is independent of the procedure used to isolate
DNA. Peaks II and III have been identified as (+~-BaP DE I-deoxy-
guanosine and BaP DE II-deoxyguanosine adducts, respectively.6 Peak
I is probably generated from 9-hydroxy-BaP, although it could be a BaP
DE I-deoxycytosine adduct. Small, late-eluting peaks were con-
sistently observed, especially in lung samples (Figure 5-6~. They
could be BaP DE I-deoxyadenosine or BaP 4,5-oxide adducts. Similar
BaP metabolite-DNA adduct profiles were observed in lung, liver, and
forestomach from ICR/Ha and C57BL/6J mice after oral administration of
BaP.9~90 Eastman _ al.51 examined the in viva binding of BaP to
DNA in lung,-liver, and kidney of Aroclor 1254-treated A/J mice after
intravenous administration of BaP. The only identified adducts
observed by Sephadex LH20 chromatography were BaP DE-DNA adducts.
Early-eluting radioactivity was present in the chromatograph.51
Eastman and Bresnick50 and Eastman et al.51 used Sephadex
LH20 chromatography to analyze the 3-MC metabolite-DNA adduct profile
in lung and liver of several mouse strains after intravenous admin-
istration of 3-MC (Figure S-7~. Two major 3-MC-deoxyribonucleoside
adduct peaks were observed in Lung and liver of each mouse strain
examined. HPLC analysis49 demonstrated seven 3-MC metabolite-DNA
adduct peaks in lung and liver of C57BL/6J mice, with the two major
adduct peaks corresponding to those observed by Sephadex LH20 chroma-
tography.50351 Early-eluting peaks (Figure 5-7) were also present in
the chromatography of these studies with 3-MC.
Binding of 3-MC, BaP, and DMBA to DNA has been examined in
skin of several mouse strains (Table 5-1~. In each study with BaP,
the major adduct observed was BaP DE-deoxyguanosine. Sephadex LH20
chromatography revealed only one adduct peak. The HPLC adduct profile
in skin was virtually identical with that in Figure 5-6 for lung,
5-18
~ . .. . ..
OCR for page 215
forestomach, and liver.ll~l4'l07,l49 The adduct profile for 3-MC
in mouse skin was the same in each strain examined, but was slightly
different from that for lung and liver (Figure 5-7), in that three
3-MC metabolite-deoxyribonucleoside peaks were observed in the
Sephadex LH20 chromatograph 2 f skin whereas only two peaks were
observed for lung and liver. 9-51,130 Sephadex LH20 chromatography
of DMBA-deoxyribonucleoside adducts in skin was similar in each mouse
strain studied; three peaks were observed.150 Early-eluting peaks
were also observed in the chromatography of these investigations of PAR
metabolite-DNA adduct formation in mouse skin.
The formation of DNA adducts of the carcinogen 15,16-dihydro-
11-methylcyclopentataiphenanthrene-17-one (11-me2t3~1ketone) was
examined in liver, lung, and skin of TO mice.l, ~ HPLC analysis
revealed eight 11-methylketone metabolite-DNA adduct peaks in each of
the tissues.2 The major adduct was generated from the interaction
between the anti-3,4-dihydro-3,4-trans-dihydroxy-1,2-dihydro-1,2-
epoxide (diol-egoxide) metabolite of 11-methylketone and deoxy-
guanosine.l,2,3 There is no major qualitative difference in the
adduct pattern among the three tissues. The adduct profile for each of
the three tissues was not substantially altered by the route of
administration (intramuscular, topical, and intraperitoneal).
In the studies with mice, the PAH metabolite-DNA binding profiles
are very similar in all tissues of all strains examined. For BaP, the
predominant characterized adduct is the BaP DE I-deoxyguanosine
adduct. When HPEC analysis was used, a BaP DE II-DNA adduct was also
observed, as well as an adduct probably generated from 9-hydroxy-BaP.
There is also evidence of BaP DE-deoxyadenosine adducts, although in
relatively small amounts. The DNA adduct profiles for 3-MC are the
same in lung and liver of each mouse strain examined and only slightly
different in skin. The pattern of DMBA metabolite-DNA adducts in skin
is the same for all strains examined. The HPLC profiles for 11-methyl-
ketone metabolite-DNA adducts are very similar in lung, liver, and
skin, with the major adduct being a diol-epoxide metabolite-
deoxyguanosine adduct.
The in vivo formation of BaP metabolite-DNA adducts has recently
been examined in male Sprague-Dawley rats and male New Zealand rabbits
(Table 5-1~. In rats, BaP was administered intravenously at 1.0 and
10.0 ~mol/kg. Several chromatographically distinct nucleoside-bound
adducts were3Observed in lung, whereas only one adduct was apparent in
the liver.2 The predominant BaP-nucleoside adduct formed in vivo
in rat lung and liver was chromatographically identical with adducts
formed on further metabolism of BaP phenols, possibly because of the
interaction of 9-hydroxy-BaP 4,5-oxide with DNA.6312323 The BaP DE
adducts were not detected in rat liver, and only a relatively small
amount was observed in rat lung. The BaP DE adducts in rat lung
accounted for only 1.4% of total UNA binding and 3.3% of the adducts
generated by BaP phenol~s). Thus, the in vivo BaP metabolite-DNA
adduct proftles obtained in lung and liver of Sprague-Dawley rats are
5-19
OCR for page 216
distinctly different from those observed in various mouse strains.
This is the only known case in which the BaP DE adduct is not the
predominant BaP metabolite-DNA adduct formed in viva.
In an examination of BaP metabolite-DNA adduct profiles in lung
liver of male New Zealand rabbits, BaP was administered either orally
or intraperitoneally at 50 mg/kg. The DNA adduct profiles were
identical with those in mice (Figure 5-6), with one notable difference
(Bixler and Anderson, unpublished data): In rabbits, there was
approximately 757 as much BaP DE II-deoxyguanosine adduct as BaP DE I
adduct, whereas in mice, there was only 10% as much BaP DE II adduct as
BaP DE I adduct. This is the only known case in viva in which the BaP
DE II-deoxyguanosine adduct approaches the BaP DE I adduct in amount.
and
It should be emphasized that, in these investigations of PAH
metabolite-DNA adduct formation, large amounts of the UNA-associated
-radioactivity chromatographed not as nucleoside-bound adducts, but
rather as uncharacterized fast-eluting peaks (Figures 5-6 and 5-7~.
Although Adriaenssens et al.3 showed that isolation of DNA by a
hydroxylapatite procedure, instead of by the precipitation method,
significantly reduces the amounts of these early-eluting peaks, the
peaks still account for a large proportion (especially in liver and
forestomach) of the total radioactivity eluted in chromatography. Some
workers have ignored these early-eluting peaks by a pre-elution step
with Sephadex LH20 chromatography (e.g., Figures 5-6 and 5-7~. These
peaks are also observed in in vitro studies and in in viva model
systems (see Boroujerdi et al.2 ). The radioactivity appears to
reflect some tissue-specific reactions, such as those exhibited by the
different patterns in lung and liver.50351 Eastman and Bresnick50
showed, by using borate-eluted Sephadex LH20 and DEAE-Sephadex
chromatography, that early-eluting radioactivity contains numerous
constituents. Studies that used [14C]BaP and [3H]BaP5~14l~l49
and the results of Eastman and Bresnick50 and Eastman et al.51
suggested that only a small amount of this radioactivity Is due to
tritium exchange, whereas experiments of other investigatorsl5~159
suggested the opposite. The results of Eastman and Bresnick50
suggested that only a small amount of the radioactivity in the early
peaks is related to oligonucleotides. Phosphoi~i~sters might
contribute to the early-eluting radioactivity. ~ 1 In any case,
because a considerable amount of radioactivity appears in the early-
eluting peaks, their identification deserves further consideration.
COMPARISON OF EXTENT OF PAH METABOLITE-DNA ADDUCT FORMATION BETWEEN
TISSUES AND BETWEEN SPECIES
_ . _
Specific activities (SAs), in picomoles per milligram of DNA, of
PAH metabolite-DNA adducts have been determined in several tissues
after administration of PAHs. Table 5-2 gives the SAs of BaP DE
adducts in lung and liver of various mouse strains and New Zealand
rabbits. The amounts of BaP DE adducts are very similar in lung and
5-20
~ . ... ..
OCR for page 217
liver for each study reported in Table 5-2. Anderson et al.8
examined the BaP DE adducts in lung, liver, and forestomach of A/HeJ
mice for oral administration of BaP at 2-1,350 ~mol/kg. The SAs of the
BaP DE adducts in lung and liver were similar over the entire dose
range and ranged over 3 orders of magnitude in this study. The SA of
BaP DE adducts in forestomach of mice is very similar to that in lung
and liver after oral administration of BaP.338390 The-similarity of
the BaP DE adduct amounts in lung, liver, and forestomach is rather
surprising, inasmuch as the disposition and rate of metabolism of BaP
in these tissues are probably very dissimilar. The higher rate of BaP
metabolism in liver is reflected in the greater total DNA binding and
protein binding in liver, compared with lung and forestomach.8
The SAs of the BaP phenol-oxide adduct were also very similar in
lung and liver of the Sprague-Dawley rat for each intravenous dose of
BaP (Table 5-3~. As mentioned previously, this was the predominant
adduct observed in lung and liver of this species.23 As with mice,
the total DNA binding was significantly higher in liver.
Eastman and Bresnick50 examined 3-MC metabolite-DNA adduct
formation (Figure 5-7) in lung and liver of several mouse strains at
several points after intravenous injection of 3-MC (12.6 mg/mouse).
Mixed-function oxidases were induced with Aroclor 1254 24 h before
injection of 3-MC. The amounts of adducts were significantly higher in
lung than in liver in each mouse strain and at each time (Table 5-4~.
Total DNA-associated radioactivity in liver is not significantly
different from that observed in lung. Thus, the relative binding of
3-MC to DNA of lung and liver of Aroclor 1254-treated mice is
distinctly different from that of BaP in untreated mice. The ratio of
3-MC-DNA binding in liver to that in lung is smaller than the ratio for
BaP for both nucleoside-bound adducts and total DNA-associated
radioactivity (Tables 5-3 and S-43. At present, BaP and 3-MC are the
only PAHs for which the amounts of PAR metabolite-DNA adducts can be
compared between lung and liver.
It should be emphasized that the SAs for the in viva studies
reported in Table 5-1 are calculated on the basis of the total DNA in
the organ. These values for the BaP DE adducts (Table S-2) do not
differentiate between lung and liver and therefore do not appear to
offer any explanation for susceptibility of the lung and resistance of
the liver to BaP-induced neoplasia in, for example, A/HeJ and A/J
mice. However, it is likely that the amounts of adducts formed in
different cell types vary considerably. This possibility has the
greatest implication for organs, such as the lung, that contain a
multitude of cell types. Although little is known about the
localization of carcinogen-DNA adducts in lung, cytochrome
P-450-dependent monooxygengse enzymes appear to be much more localized
in lung than in liver.4~'lb5,l06 The conciliated bronchiolar
epithelial (Clara) cells of rabbit lung have been identified as having
high concentrations of these enzymes--a finding that correlates with
the observed pulmonary toxicity of 4-ipomeanol, which is thought to
5-21
Hi;
- . ,i, . ...
OCR for page 262
192. Yamaura, I., B. H. Rosenberg, and L. F. Cavalieri. The major
adducts of cis and bans benzo [a] pyrene diol epoxides cause
chain termination during DNA synthesis in vitro. Chem. Biol.
Interact. 37: 171-180, 1981.
193. Yang, L. L., V. M. Maher, and J. J. McCormick. Error-free
excision of the cytotoxic and mutagenic N2-deoxyguanosine
DNA adduct formed in human fibroblasts by (+~-7S,8a-dihydroxy-
9~,10~-epoxy-7 ,-8 ,19, 10-tetrahydrobenzo [a] pyrene. Proc. Natl.
Acad. Sci. USA 77: 5933-5937, 1980.
194. Yang, S. K., P. P. Roller, and H. V. Gelboin. Benzo[a]pyrene
metabolism: Mechanism in the formation of epoxides, phenols,
dihydrodiols, and the 7,8-diol-9,10-epoxides, pp. 285-301. In
P. W. Jones and R. I. Freudenthal, Eds. Carcinogenesis--A
Comprehensive Survey. Vol. 3. Polynuclear Aromatic Hydro-
carbons: Second International Symposium on Analysis, Chemistry
and Biology. New York: Raven Press, 1978.
5-66
=. .. .. ..
OCR for page 263
6
POLYCYCLIC AROMATIC HYDROCARBONS IN FOOD AND WATER
AND THEIR METABOLISM BY HUMAN TISSUES
This chapter deals with the relation of PAHs to human metabolism.
Specifically, its purposes are to collate a large volume of literature
dealing with the capacities of a number of human tissues to interact
with and biotransform selected PAHs; to define, where possible, the
effects of these compounds on human tissues; and to examine the
principal sources of human exposure to PAHs through food and water.
PAR METABOLISM BY HUMAN TISSUES
The abilities of various human tissues to metabolize PAHs have been
extensively studied, with emphasis on the chemical biotransformations
that are catalyzed by tissues that can be readily sampled (such as
blood cells, skin, and placenta) or that can be biopsied or cultured
(such as fibroblasts, liver, and intestinal and tracheohronchial
epithelium). The chemical biotransformations of selected PAHs that
such tissues carry out are in general qualitatively similar to those
demonstrated in animal tissues, although there are considerable species
and organ differences in catalytic activities of relevant enzymes.
These differences may be great enough to preclude comparative
generalizations; and for the most part the relation between in vitro
and in vivo enzymatic activities is unclear. Moreover, it is apparent
from the findings reviewed in this chapter that the enzymatic capacity
to biotransform PAHs to ultimate carcinogens in various tissues is not
necessarily correlated with the demonstrated ability of PAHs to produce
cancers in those tissues.
SKIN
That benzota]pyrene hydroxylase can be induced in cultured human
skin was first demonstrated in l972.1° Foreskins from children who
were circumcised 2-4 d after birth were shown to contain an enzyme that
bydroxylates the carcinogen benzo~aipyrene (BaP), and induction of the
enzyme (by a factor of 2-5) was demonstrated when the foreskins were
cultured for 16 ~ in the presence of 10 AM henz~ajanthracene. Among a
group of 13 skin samples studied, control enzymatic activities extended
over a threefold range and were not correlated with race, age of
mother, or medications given to mother or infant.2 The enzyme had an
absolute requirement for NADER and molecular oxygen and was completely
inhibited by CO; these findings suggested the involvement of a species
of cytochrome P-450 in the bydroxylation reaction. The presence of
this heme protein in low concentrations in cutaneous tissue was later
6-1
At;
. ~. ... .
OCR for page 264
demonstrated by Bickers et al.22 Coal-tar products, which are widely
used in dermatologic practice in conjunction with exposure to
ultraviolet light (~.g., the Goeckerman regimen for psoriasis62963),
were also shown to induce aryI hydrocarbon hydroxylase (AHH) signifi-
cantly in patients with dermatologic disease where the coal tar was
applied, but not in skin distant from the site of application.21 In
concurrent studies in neonatal rats,21 although (as in humans)
distant skin sites were unaffected by the coal-tar application, AHH
activity in the livers of treated animals increased to more than 20
times the c,ytto] values. Among five identifiable constituents of coal
tar studied for their AHH-inducing properties in human skin, BaP
was the most potent; pyrene and anthracene also caused significant
induction of the enzyme. In isolated cultured human hair follicles,
Vermorken _ al.,185 using radioactive BaP as substrate, not only
demonstrated the presence of the hydroxylase, but identified the
formation of the 3-OH, 7,8-dihydro-7,8-dihydroxy, and 9,10-dihydro-
9,10-dihydroxy metabolites of this PAH.
BaP clearly has cytotoxic effects on cultured human skin
fibroblasts, although relatively high concentrations are required for
cytotoxicity. Milo et al.123 studied the influence of the three
carcinogenic PAHs, 7,12-dimethylbenzanthracene (7,12-DMBA), 3-methyl-
cholanthrene (3-MC), and BaP on mixed-function oxidase activity,
cell proliferation kinetics, and DNA damage in cultured fibroblasts.
They found that only BaP, at 10 ~g/ml or higher, affected all the
cellular metabolic characteristics examined. 7,12-DMBA at 6 ~g/ml or
higher induced the mixed-function oxidase system and stimulated DNA
synthesis; 3-MC at concentrations as high as l5 ~g/ml produced no
significant cellular alterations. Similarly, 5-fluoro-7,12-DMBA,
anthracene, and phenanthrene had no effects on these buman cells. The
authors concluded that BaP alone could initiate all the biochemical
events probably necessary to trigger transformation of human cells in
v~tro.
PAH-induced cytotoxicity to cultured human fibroblasts has also
been demonstrated by Strniste and Brakel72 and Aust et al.8 In the
former study, normal fibrohlasts and xeroderma pigmentosum (XP) cells
were used, and BaP was "activated" by light radiation (near
ultraviolet), rather than enzymes. Photoactivation (at 300-400 nm) of
BaP produced at least three identifiable quinones (1,6-, 3,6-, and
6,12- isomers), as well as more hydrophilic products, depending on the
duration of light exposure. Formation of these products was
oxygen-dependent. The irradiation products led to several types of ONA
*"Mixed-function oxidase" refers to the NADPH-dependent enzyme complex
containing cytochrome P-450s in the membranes of the endoplasmic
reticulum, which catalyzes the oxidation of numerous structurally
diverse molecules, including drugs, steroid hormones, and carcinogens.
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damage, with covalently bound hydrocarbons constituting the major
lesion under all conditions studied. XP cells were more sensitive to
damage than normal cells (by a factor of 1.7-2), and sensitivity
increased by a factor of 10 when long-wavelength ultraviolet light was
used. 7,12-D~BA, 3-MC, and BaP were also examined; the order of
phototoxicity was 7,12-DMBA > BaP > benzote~pyrene > 3-MC.
In the study of Aust et al.,8 a human epithelial cell-mediated
cytotoxicity and mutagenicity assay system for BaP was developed with
human fibroblasts as the target cells. Lethally x-irradiated human
kidney-carcinoma epithelial cells were cocultivated with human XP skin
fibroblasts (XP12BE) lacking excision-repair capability for BaP-DNA
adducts. Under defined conditions, the frequency of mutation to
6-thioguanine resistance and PAH binding to DNA were shown to be
concentration-dependent. Two principal BaP-DNA adduct peaks could be
identified--a major peak consistent with an adduct standard synthesized
from the anti-isomeric 7,8-dibydrodiol-9,10-epoxide of the hydrocarbon
and a minor peak consistent with the syn-isomeric form of this
metabolize. The results are consistent with those in other reports on
BaP adducts formed in human explant tissue from lung,161 colon,l°
esophagus,68 and bronchus,75 and they represent an advance in the
development of sensitive assay systems for detecting biologic responses
to human epithelial-cell activation of BaP.
Direct neoplastic transformation of human fibroblasts by
carcinogens has also' been demonstrated. In the study of Kakunaga,79
normal human adult fibroblasts exposed to the carcinogen 4-nitro-
quinoline' 1-oxide underwent malignant transformation In a process
requiring numerous cell divisions. When injected into athymic (nude)
mice, the transformed cells produced solid tumors at the site of
inoculation. Because it could not be metabolically activated by the
target cells used, 3-MC was unable to effect transformation; the use of
other PAHs and induced microsomes with high concentrations of
cytochrome P-450 to activate 3-MC was not examined. Normal human
foreskin cell populations were neoplastically transformed in studies by
Milo and DiPaolo 24~125 with a number of non-PAH carcinogens; and
treatment with a tumor promoter alone (phorbol ester) has been
shown90 to induce neoplas tic transformation' in fibroblasts from
humans genetically predisposed to cancer (familial polyposis of the
colon). Thus' it can be inferred that cells already in an "initiated
state" as a result of a genetic defect represent a novel fibroblast
system that may provide a means for exploring separately the roles of
initiators and promoters in carcinogenesis. Painterl35 used HeLa
cells to develop a rapid screening test to detect agents that damage
human DNA. The test measures thymidine uptake into the cells at
various times (principally 1-2 h) after treatment with a presumptive
carcinogen or mutagen. In this test system, BaP was inert unless
metabolically activated by incubation with rat-liver microsomes.
Brookes and Duncan26 compared the effects of PAHs on primary human
embryo cells and HeLa cells. Fibroblasts from skin, lung, muscle, and
gut were cultured and treated with 3H-labeled BaP and 7,12-DMBA.
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Both hydrocarbons were metabolized in the cultures, 7,12-DMBA more
slowly than BaP; among the cell types studied, lung fibroblasts
metabolized the compounds more efficiently than others and retained
this capacity well in subculture. The binding of BaP and 7,12-DMBA to
DNA, RNA, and protein of these primary lung-derived fibroblasts was
also studied (metabolism of each hydrocarbon exceeded 75% during the
48-96 h of treatment) and was found to be significantly greater for BaP
than for 7,12-DMBA. The data in this study also established
parallelisms, at least for BaP, between hydrocarbon binding to cellular
macromolecules in fibroblast cultures derived from mouse embryos and
those derived from human lung cells. Such parallelism is of more than
casual interest, in view of the susceptibility of the mouse to
hydrocarbon carcinogenesis and the known correlation between
hydrocarbon-DNA binding and cancer-producing activity in mouse skin.
The effects of pyrene and BaP in the human diploid fibroblast
culture WI-38 were studied by Weinstein et al.188 Neither caused
significant damage (compared with controls), as assessed by mitotic
index or chromosomal breaks after 1-h pulse exposures. However,
metabolic activation of BaP with microsomes resulted in a dramatic
decrease in mitotic index and a significant increase in breakage.
Microsomal incubation did not alter the inertness of pyrene in this
test system. Freeman et al.60 have made interesting observations on
comparative aspects of hydrocarbon metabolism in skin epithelial and
fibroblast cultures. A comparison of the ability of epithelial-cell
colonies and of fibroblast colonies from the same 13 subjects to
metabolize BaP to a water-soluble form demonstrated clearly the
markedly greater metabolizing capacity of epithelial cells. There was
a 20-fold difference in this capacity of epithelial cells; within
individual subjects, the ability of epithelial cells to metabolize the
PAN exceeded that of fibroblasts by as much as a factor of 40. There
appeared to be a major effect of culture age (6-55 d) on the ability of
epithelial cells to metabolize BaP.
A direct toxicity of BaP to normal human epithelial-cell cultures
has been described by Dietz and Flaxman.5O This toxicity was
reflected in a dramatic reduction in epidermal-cell outgrowth, a
decrease in mitotic indexes, a loss of the well-ordered cell
relationships, and the early appearance of giant cells ranging in
diameter from 100 to 200 ym. In a clinical study that clearly could
not be carried out today, Cottini and Mazzone45 (in 1939) applied a
1% solution of BaP daily (up to 120 d) to the skin of 26 normal
subjects and patients with various dermatologic disorders and examined
the gross and histologic consequences. The sequential epidermal
changes, of which gross pigmentation and verrucae were the most
frequent, and histologic alterations (which regressed within several
months when treatment was terminated) led the authors to conclude that
"benzopyrene, if applied to human skin for protracted periods, would be'
carcinogenic as it is in animals."
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The metabolism of benz~ajanthracene, 7,12-DMBA, and BaP by human
mammary epithelial-cell aggregates in culture has been investigated by
Grover et al.66 with nonneoplastic tissue obtained from eight
patients undergoing reduction mammoplasty. All three PAHs were
metabolized to water-soluble and organic-solvent-soluble products; the
latter included K-region and non-K-region dihydrodiols. The major
dihydrodiols detected as metabolites of the parent PAHs were the
8,9-dihydrodiols of BaP and 7,12-DMBA and the 9,10-dihydrodiol of BaP.
The hydrocarbons bound to the proteins and DNA of the epithelial cells,
but there were wide differences between different PAHs in extent of
binding between tissue preparations from different patients. Some of
the PAH-deoxyribonucleoside adducts formed from 7,12-DMBA and BaP
appeared to have been produced through reactions of bay-region
diol-epoxides with DNA, but little reaction with DNA was detected in
tissue preparations treated with BaP.
Unscheduled DNA synthesis induced by DNA-damaging chemicals has
been measured in nonreplicating human fibroblasts by autoradiographic
methods that are not readily applicable to organotypic epithelial-cell
cultures. To evaluate the range of chemical sensitivity and DNA-repair
responses of human skin epithelial cells, Lake et al.95 developed a
semiquantitative in vitro method for measuring unscheduled DNA
synthesis in normal foreskin epithelial cells. On serial subculture of
organotypic primary skin cultures, the unscheduled DNA synthesis
response elicited by 3-MC decreased in parallel with the ability of
cells to metabolize PAHs to water-soluble metabolizes. The working
hypothesis was that procarcinogens that are efficiently activated by
human skin-specific metabolism will be detected with unscheduled DNA
synthesis as an end point.
LIVER AND INTESTINE
l
Obana et al.l30 analyzed quantitatively and qualitatively the PAH
content of samples of human liver and fatty tissue. Six samples of
liver and 10 samples of fat were obtained at autopsy from 10 persons
who died of unstated causes (although the tissues were reported to be
"free from cancers. Smoking habits, occupations, etc., were not
described. The tissue samples analyzed were quite large (40-120 g),
and the PAHs were determined without complex pretreatment. Table 6-1
shows the analytic results for liver, and Table 6-2 the comparable data
for fat tissue. Note that PAH concentrations are expressed as parts
per trillion (ppt), not parts per billion (ppb), and are in general
extremely low. Nevertheless, the data indicate that, on the average,
the PAH concentration in liver was one-third that in fat. Pyrene had
t'ne highest concentration, followed by anthracene. Although the number
of samples was small, no sex or age differences were evident. The
known carcinogens benz~ajanthracene and dibenz~ah~anthracene were not
detected in either tissue. However, BaP was detected in small amounts
(20 ppt) in both liver and fat. This finding should be compared with
that of Tomingas et al.,178 who detected BaP at 1-15,000 ppb in
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human bronchial-carcinoma tissue (24 samples). Obana et al.130
called attention to the fact that the PAHs in the human tissues they
examined were different, in both concentration and composition, from
the PAHs that had been identified in marine samples. For example,
pyrene was found in oysters at 7-52 ppb and in Wakame seaweed at 12-41
ppb; the comparable figures for BaP were 0.3-2.6 ppb and 0.6-9 ppb,
respectively. Mor'eover, although pyrene was the most abundant PAH in
all cases, the next most abundant in the human tissues was anthracene,
whereas in the marine samples the next most abundant were
benzoteipyrene and benzotbifluoranthene. These qualitative and
quantitative distinctions, especially the marked concentration
differences between nontumorous130 and tumorous tissues178 and
between a common food source in the area and the human tissue samples,
need to be recalled in evaluating the importance of the food content of
PAHs, as well as the role of malignant pathology, when trying to
determine the significance of the body or tissue burden of these
hydrocarbons.
The liver contains the highest concentration of cytochrome P-450 in
the body. The activity of the pathway by which heme, the prosthetic
group of this heme protein, is synthesized can' tee greatly induced by a
host of foreign chemicals and can approach the rates of heme synthesis
in erythroid cells; and the enzymatic capacity of hepatic cells to
carry out the biotransformations that characterize the great variety of
PAH metabolites formed in vitro, and probably in viva, has been well
defined. Only selected PAH transformations catalyzed by liver cells
are reviewed here, with some emphasis on the relationships of PAH- 'and
drug-metabolizing capabilities and on recent data indicating that
carcinogen metabolism may be increased by direct actions on relevant
membrane-bound enzymes, as well as by the conventionally assumed
process of increased de nova synthesis of enzyme protein, i.e.,
Induction.
Dybing et al.54 have examined the in vitro metabolism and
metabolic activation of several carcinogenic PAHs in subcellular
fractions from seven human livers. The patients all suffered from
total cerebral infarction and were serving as potential kidney donors
(maintained temporarily by life-support systems) at the Huddinge
University Hospital in Sweden. At the appropriate time, liver
extirpation was perfo..~`ed; within 20 min after the procedure, perfusion
had been completed and the tissue frozen in liquid nitrogen. This
study may mimic the enzymatic properties of human liver cells in the
living subject as closely as experimentally possible, other than by
direct biopsy or surgery in a living patient. Because of the unique
source of the tissue studied, some of the data merit recording here.
Microsomal cytochrome P-450 content (seven livers) was 0.16-0.60
nmol/mg of protein, with a mean + S.D. of 0.36 + 0.15, and ASH activity
averaged 175 + 138 pmol/mg of protein per minute; one sample had a
value of 483 pmol/mg. These activities are approximately the same as
those in liver microsomes of untreated mice and rats. ASH activities
expressed per nanomole of cytochrome P-450 varied by a factor of 2.8
among the seven liver samples.
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Conney and co-workers27~28)4o,4l,73~8l,82~94,l56,l57,l74
conducted a series of studies of direct liver-cell metabolism of
carcinogens and compared such metabolism with that of drugs. They
established that carcinogen metabolism may be increased not only
through enzyme induction, but through enzyme activation as well. They
compared the oxidative metabolism of BaP with that of antipyrine,
hexobarbital, coumarin, zoxazolamine, and 7-ethoxycoumarin in 32 adult
liver samples obtained at autopsy some 8-20 h after death. When enzyme
activity for one substrate was plotted against enzyme activity for a
second substrate for each of the 32 liver samples, significant
correlations were found. For example, for BaP paired against anti-
pyrine, hexobarbital, zoxazolamine, and coumarin, the correlation
coefficients were 0.85, 0.72, 0.69, and 0.57, respectively. Some
drug-drug metabolizing activities also showed a high correlation (e.g.,
antipyrine and hexabarbital, r = 0.79; antipyrine and coumarin, r =
0.72), whereas in other instances, metabolizing capacities did not have
a high correlation, e.g., carcinogen vs. drug (BaP and
7-ethoxycoumarin, r = 0.35) and drug vs. drug (e.g., hexobarbital and
7-ethoxycoumarin, r ~ 0.37~. The findings raise the possibility that
an in viva drug-metabolism assay (e.g., a plasma disappearance-rate
study of a suitable test drug) might predict some carcinogen-
metabolizing capabilities of a person and suggest the presence in
humans of multiple monooxygenase systems for the substrates studied, as
well as their heterogeneous distribution in the population. Individual
differences in the rates of metabolism of BaP (7-fold) in these and
other liver samples studied41 were considerable, although they did
not approach the known species differences40 in rates of metabolism
of drugs.
The effects of PAH administration on the metabolic disposition of
specific carcinogens such as BaP, have not been studied in humans, but
Schlede et al.15 ,1 7 recently examined the metabolic disposition of
radiolabeled BaP in rats, and the results probably can be extrapolated
to humans. Pretreatment of rats with unlabeled BaP greatly 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 dose of the radiolabeled material, and the
increased rate lasted for at least 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 varied results
were observed in lung tissue . These influences of BaP pretreatment on
a later intravenous dose of the 3H-labeled chemical were also
observed when the radiolabeled PAH was administered orally. 3-MC and
7,12-DMBA pretreatment of animals produced comparable effects on the
metabolic disposition and tissue contents of radiolabeled BaP. Pyrene,
anthracene, and phenobarbital had little or no such effect on the in
viva disposition of this compound. In other studies, the biliary
excretion of [14CiBaP was shown to be increased by pretreatment with
the unlabeled compound; however, no increase in excretion into bile of
the 14C-labeled metabolites of BaP was observed after prior
administration of this PAH. These findings suggest that conversion of
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BaP to its metabolites may be the rate-limiting step in the biliary
excretion of the compound. Phenobarbital had no effects on the pharma-
cokinetics (plasma disappearance) of [14C]BaP and its metabolites;
this drug might stimulate the conjugation of hydroxylated BaP
derivatives before their excretion into bile. The relevance of these
findings to humans is related not only to the (probably) qualitatively
similar pharmacokinetics of such a chemical as BaP--particularly its
extensive excretion via bile--but also to the potentially important use
of selected drugs, singly or multiply, to increase disposal of PAHs and
their metabolites from the body by stimulating conjugation and biliary
excretion and by increasing otherwise innocuous metabolic
biotransformations.
Prough et al. 143 have also studied BaP metabolism in human liver,
kidney, and lung, and they characterized the metabolites formed by HPLC
techniques. Tissue samples were obtained within 1-5 h after death, and
assays were completed within the succeeding 2-4 h. In the analysis of
metabolites formed from BaP, quinones, three classes of dihydrodiols,
and two classes of phenols were categorized. Table 6-3 summarizes the
rates of formation of these metabolites by tumor, liver, kidney, and
lung microsomes and presents comparable data in rodents. There was a
very large variation in the human metabolism of BaP, compared with that
demonstrated in studies carried out concurrently in rodents. That
might reflect, as the authors noted, either the controlled environment
of the animals studied or a genetic variation in humans. In the human
liver, activities for metabolite formation were substantially lower
than those in rat microsomal fractions, and there were significant
differences in the BaP-metabolite profiles. A greater proportion of
benzene-ring metabolites was formed by human lung microsomes than by
human liver and kidney microsomes (or rodent lung microsomes). The
relative increase in the 9,10-dihydrodiol product, as well as some
increase in the 7,8-dibydrodiol metabolite, accounted for the larger
portion of this difference among lung, liver, and kidney microsomes.
There is an apparent biologic inconsistency in these findings: although
the human lung is the principal site of PAR tumorigenesis, as the
authors observed, the 9,10-dihydrodiol product is suggested to have
little biologic activity on further metabolism, whereas other tissues,
such as the liver, formed large concentrations of the 7,8-dihydrodiol,
a "proximate carcinogen.'' Nevertheless, the findings are important,
providing not only data on comparative rates of formation of metabo-
lites constituting the "HPLC profiles" in man and rodents, but also
intertissue metabolic profiles of BaP biotransformation for three major
organ systems in man. Thus, they extended the earlier findings of
Selkirk et al.160 on liver cells and lymphocytes.
A major advance in defining the role of the liver in PAN metabolism
and the factors that regulate liver monooxygenase activity has been the
demonstration that hepatic microsomal oxidation of specific substrates
can be directly increased in vitro, in addition to their DroDerty of
being induced in viva, by various chemical treatments.27~28~73~174
Conney and colleagues have shown that 7, 8-benzof lavone added to
homogenates of human liver samples can increase the rate of BaP
6-8
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hydroxylation by a factor of up to 11. Benzoflavone also increased
some drug hydroxylations substantially, although benzoflavone at
concentrations lower by a factor of about 100 paradoxically inhibited
these reactions. Marked individuality for activating and inhibiting
effects of benzoflavone were noted, and no significant effects on the
oxidation of some drug substrates (e.g., coumarin and hexobarbital)
were observed.
The enhancing effect of 7,8-benzoflavone on BaP oxidation was shown
to extend to o ther f lavonoids, such as f lavone, nob i le t in, and
tangeritin. Related compounds--such as apigenin, chrysin, fisetin,
flavonone, galargin, hesperitin, kaempferal, morin, myricitin,
naringerin, and quercetin--inhibited BaP oxidation. The stimulatory
effect of 7,8-benzoflavone on BaP 9,10-dihydrodiol oxidation to
bay-region epoxides was also studied and shown to have significant
species-specific characteristics. With untreated hamster microsomes,
more than 60% of the total metabolites of the hydrocarbon were
bay-region diol-epoxides, whereas human liver formed less than it of
such metabolites. Addition of 7,8-benzoflavone to the microsomal
incubations dramatically stimulated the formation of these metabolites
in human (and rabbit) liver microsomes. The stimulatory effects of
flavonoids on hydrocarbon oxidation have recently been shown to occur
in vivo as well,l°° so the biologic importance of this phenomenon for
the intact host is likely to be considerable. The mechanism of
flavonoid activation of BaP hydroxylation has recently been explored in
detail by Huang et al.73 The flavone stimulates the NADPH reduction
of cytochrome P-450, but not that of cytochrome c, by NADPH-cytochrome
c reductase; this finding supports the idea that the catalytic sites
for these substrates of the reductase are different. Other evidence
that these catalytic sites are different has also recently been
provided by the studies of Yoshinaga et al.197-200
Metabolic transformation of PAHs and their binding to cellular
macromolecules in cultured human gut tissues have been described.
Harris et al.68 examined the metabolic fate of BaP and several other
compounds in cultured esophogeal explants from eight patients, six of
whom had esophageal carcinomas. Metabolism of the 3H-labeled PAH to
water-soluble ~etabolites varied among the eight patients over the
range of 1-68% of total metabolism; the variation found within a single
case, however, in relation to different anatomic segments of the
esophagus (proximal, middle, and distal) was quite narrow--2%. In
spite of the 68-fold variation in metabolism among subjects, the
patterns of conjugates formed from metabolites in general were
qualitatively similar: sulfate esters, 21-55%; glucuronide conjugates,
7-37%; and glutathione conjugates, 24-66%. Most of the radioactivity
of the organic-solvent-soluble metabol ites of BaP cochromatographed
with authentic metabolites of this compound, including its proximate
carcinogenic, (-~-trans-7 , 8-dihydro-7,8-dihydroxy, derivative. Despite
the predominant occurrence of esophageal cancer in the distal segment,
the patterns of metabolites formed in all segments of the organ were
similar. Binding of 3H-labeled PAHs to ONA and protein was detected
in all eight cases, with binding to protein greater than to ONA in each
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instance. Binding among the eight cases varied 99-fold, and at least
three hydrocarbon-DNA adducts, including the specific guanine adduct,
were recognized. The adducts appeared identical with those previously
found in human colon and bronchus; and the patterns of both metabolism
and adduct formation with BaP were analogous to those found in
experimental animals susceptible to the carcinogenic action of PAHs.
Autrup et al.,9 in an earlier and less detailed but essentially
similar study, had reported comparable data based on human colon
explants of tumor-free tissue. The binding of labeled BaP to cellular
protein was several times higher than that to DNA; however, hydrocarbon
binding to DNA correlated with tissue AHH activity (r = 0.87), whereas
no such correlation existed for protein binding. DNA binding (BaP,
picomoles bound per 10 ma) among seven tissue samples varied over a
25-fold range; variation within subjects was small. In an extension of
this work, Autrup et al.ll studied the comparative metabolism of BaP
(and aflatoxin B1) and hydrocarbon-DNA adduct formation in cultured
human and rat colon explants. Adduct formation (in 103 cases) varied
over a 125-fold range in the tumor samples and in the same subject over
a 3- to 10-foId range in different segments of the organ. A number of
hydrocarbon-DNA adducts were identified, and both qualitative and
quantitative rat-human differences were demonstrated. Although overall
BaP metabolism was similar in rat and human colon tissue, the ratio of
organic-solvent-soluble to water-soluble metabolites was higher in the
human; sulfate esters predominated in rat colon, whereas equivalent
quantities of sulfate esters and glutathione conjugates were formed in
the human tissue; and hydrocarbon-DNA binding was distinctly greater in
human colon, although, as noted, there was marked variability in adduct
formation within a given subject.
The comparative hydration of styrene 7,8-oxide, octane 1,2-oxide,
naphthalene l, 2-oxide, phenanthrene 9,10-oxide ? benz~aJanthracene
5,6-oxide, 3-MC 11,12-oxide, dibenz~ah~anthracene 5,6-oxide, and BaP
4,5-, 7,8-, 9,10-, and 11,12-oxides to their dihydrodiols was
investigated in microsomes from nine human liver samples obtained at
autopsy.80 The substrate specificity of the epoxide hydratase in
human liver ~nicrosomes was very similar to that of the epoxide
hydratase in rat liver microsomes. Phenanthrene 9,10-oxide was the
best substrate for the human and rat epoxide hydratases, and
dibenz~ah~antoracene 5,6-oxide and BaP 11,12-oxide were the poorest
substrates. Plotting epoxide hydratase activity obtained with one
substrate against epoxide hydratase activity for another substrate for
each of the nine human livers revealed excellent correlations for all
combinations of the 11 substrates studied (r = 0.87-0.99~. The data
suggest the presence in human liver of a single epoxide hydratase with
broad substrate specificity, although the results do not exclude the
possible presence in human liver of several epoxide hydratases that are
nder similar regulatory control.
6-10
Representative terms from entire chapter:
adduct amounts
