Rat and Human Expiant Metabolism

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Rat and Human Expiant Metabolism Powered By Docstoc
					[CANCER RESEARCH 45,6225-6231, December 1985]

Rat and Human Expiant Metabolism, Binding Studies, and DMA Adduct
Analysis of Benzo(a)pyrene and Its 6-Nitro Derivative1

R. Colin Garner, Caroline A. Stanton, Carl N. Martin, Curtis C. Harris, and Roland C. Grafstrom
Cancer ResearchUnit, University of York, Heslington, York Y01 5DD, United Kingdom [R. C. Ga., C. A. S., C. N. M.]; Human TissueStudies Section, Laboratory of Human
Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20205 [C. C. H.]; and Department of Toxicology, Karolinska Institute, Box
60400 S-10401, Stockholm, Sweden [R. C. Gr.]

ABSTRACT                                                                           the particular HPLC gradient used, this may indicate that the
                                                                                   adducts are structurally similar.
   Human colon and bronchus tissue expiants were incubated
with either [3H]benzo(a)pyrene ([3H]BP) or [3H]-6-nitrobenzo-
(a)pyrene ([3H]-6-NBP). The total percentage of metabolism of                        INTRODUCTION
BP and 6-NBP was, respectively, 8-59% and 18-41% in bron
                                                                                       PAH2 are ubiquitous environmental pollutants arising through
chus and 11-23% and 36-50% in colon. A product tentatively
identified as 3-hydroxy-6-NBP was isolated from the 6-NBP                            incomplete oxidation of organic matter. A large number of PAH
incubation medium. BP and 6-NBP when incubated at equivalent                         have been structurally characterized and identified as having
                                                                                     carcinogenic activity in animals. BP is present in many environ
concentrations were found to bind covalently to the DNA of
                                                                                     mental samples and has, because of its carcinogenic activity in
human bronchi from 15 cases at means of 42 and 50.9 pmol/10
                                                                                     experimental animals, been used as a measure of pollution (1).
mg DNA, respectively, and to the DNA of human colon from 6
                                                                                     The metabolism and DNA binding of BP in vitro and in vivo has
cases at means of 66.5 and 35 pmol/10 mg DNA, respectively.                          been intensively studied. The major detectable BP bound DNA
The range among individuals was within one order of magnitude.                       adduct is formed as a result of the BPDEI metabolite binding
High pressure liquid chromatography (HPLC) of enzymic hydrol-
                                                                                     through the extracyclic amino group of deoxyguanosine. This
ysates of human bronchus explant DNA revealed one adduct                             adduct has been identified in vitro in rat hepatocytes (2), hamster
from the BP-incubated bronchus which cochromatographed with                          embryo cells (3), mouse embryo cells and hamster kidney cells
(±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)py-                         (4), peripheral human lung tissue (5), human and bovine tissue
rene-deoxyguanosineand a possible two adducts from the 6-                            expiants (6-8), perfused rat liver and lung (9-11), perfused
NBP-incubated bronchus which eluted earlier than did the BP                          mouse liver and lung slices (10), and mouse skin (6,12,13). The
adduct. DNA obtained from the lung or liver of rats given 2.0-                       diol-epoxide adduct was also the major adduct detected In vivo
mg/kg doses of either [3H]BP or [3H]-6-NBP by i.p. injection was                     in female A/HeJ mice (14-16) and in C57BL/6J mice (15). The
also enzymically hydrolyzed and analyzed on HPLC. Three DNA                          major adduct isolated from the liver and lung of rats given i.v.
adducts were observed in liver and two were observed in lung                         injections of BP was not the BPDEI adduct but was thought to
DNA hydrolysates from rats given injections of [3H]BP. One                           be the result of the further metabolism of 9-OH-BP (17). BP
adduct from each organ cochromatographed with (±)-7,8-dihy-                         incubation with DNA and 3-methylcholanthrene-induced rat liver
droxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene-deoxy-                            microsomes, isolated rat liver nuclei, or 3-methylcholanthrene-
guanosine; however, the major adduct in each case eluted earlier.                    induced rat hepatocytes resulted in the formation of a major
                                                                                     product other than the BP diol-epoxide:DNA adduct which was
Only one adduct was detected in liver and lung DNA hydrolysates
from rats given [3H]-6-NBP, and this had the same retention time                     thought to be derived from the 9-OH-BP metabolite (18-20). It
as did the major adduct isolated from human bronchus that had                        would appear that, depending on the system used, there are at
been incubated previously with [3H]-6-NBP. Salmonella typhi-                         least two major pathways by which BP can be metabolized to a
murium TA98 was incubated with [3H]-6-NBP and Aroclor-in-                            DNA-binding species resulting in differences in the major DNA
                                                                                   adduct formed.
duced rat liver S9. Enzymically hydrolyzed DNA analyzed by                            Interest has recently been focused on nitrated PAH. Nitro-PAH
HPLC revealed three adducts, two of which cochromatographed
                                                                                   are readily formed following exposure to nitrogen oxide in the
with the two DNA adducts isolated from human bronchus DNA
                                                                                   test tube or, more importantly, during the combustion of diesel
adduct which had the same retention time as did the major liver
and lung DNA adduct from rats given i.p. injections of [3H]-6-                     fuel. They also may be formed during atmospheric reactions and
                                                                                   have been identified in collected air particulate samples (21 -23).
NBP. In each case the major adduct from DNA hydrolysates of                        Interest in these compounds originated from the finding that
rat liver and lung, human bronchus, and S. typhimurium, all                        almost all are potent direct-acting bacterial mutagens in the Ames
treated with [3H]-6-NBP, cochromatographed with the major
                                                                                   Salmonella assay (24) and are biologically active in several types
DNA adduct isolated from liver and lung DNA of rats given                          of mammalian cells (25-27). It is not yet certain as to how most
[3H]BP. Because BP and 6-NBP markers were not separable on
                                                                                        2The abbreviations used are: PAH, polycyclic aromatic hydrocarbons; BP,
   Received 12/19/84; revised 7/24/85; accepted 8/26/85.                             benzo(a)pyrene; BPDEI, (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzc-
   ' This investigation was supported by a grant from the Health and Safety          (a)pyrene; 9-OH-BP, 9-hydroxybenzo(a)pyrene; 7,8-diol, 7,8-dihydrodihydroxy-
Executive. C. N. M. and R. C. G. are supported by the Yorkshire Cancer Research      benzo(a)pyrene; 3-OH-BP, 3-hydroxybenzo(a)pyrene;           6-NBP, 6-nitro-
Campaign. R. C. Gr. was supported in part by the Swedish Cancer Society and          benzo(a)pyrene;3-OH-6-NBP,3-hydroxy-6-nitrobenzo(a)pyrene;dGuo, deoxygua
the NationalBoard for Laboratory Animals.R. C. Ga.was a recipientof a Yamagiwa-      nosine; HPLC, high-pressure liquid chromatography; Bis-Tris, 2,2-bis (hydroxy-
Yoshida fellowship from the International UnionAgainst Cancer.                       methyl)-2,2',2"-nitriloethanol.

                                                    CANCER      RESEARCH       VOL. 45 DECEMBER           1985
                                   BINDING STUDIES AND DNA ADDUCI                  FORMATION      BY BP AND 6-NBP

of these compounds exert their biological effects. However, in                  was redissolved in 1 ml benzene and purified on an alumina column.
                                                                                [3H]-6-NBP was eluted with benzene as a yellow band, and the solvent
the case of 1-nitropyrene, the available evidence indicates acti
vation through reduction of the nitro group to form a DMA-                      was removed under reduced pressure. The residue was redissolved in
                                                                                30% (v/v) benzene in ethanol and stored at -90°C. Radiochemical purity
binding species in both bacteria and rats (28, 29). Conversely 6-
                                                                                was determined on HPLC and the identity of the compound was con
NBP, which has been shown to bind covalently to DNA of various
                                                                                firmed by cochromatography with authentic standard prepared by the
organs when injected i.p. into rats (30), does not appear to be
                                                                                method of Fieser and Hershberg (34). UV and mass spectrum, melting
activated via the nitro group. When incubated in in vitro systems               point, and carbon-hydrogen-nitrogen analysis all confirmed the structure
such as rat microsomes, the metabolites detected included 1-                    of the compound to be 6-NBP.
and 3-OH-6-NBP, 6-NBP-1,9-hydroquinone,        6-NBP-3,9-hydro-                     Treatment of Animals with [3H]-6-NBP and [3H]BP. Male Wistar-
quinone, and BP-3,6-dione (31). These metabolites most proba                    derived rats purchased from Bantin and Kingman, Hull, United Kingdom,
bly arise from a 2,3-epoxide intermediate (31). In an intact cell               were housed two per cage, fed modified diet 41B (Oxoid Ltd., London,
                                                                                United Kingdom) and given water ad libitum. [3H]-6-NBP (2 mg/kg;
culture system such as Syrian hamster embryonic fibroblasts,
                                                                                specific activity, 1.18 Ci/mmol) or [3H]BP (2 mg/kg; specific activity, 1.26
there is some evidence for the production of dihydrodiols from
6-NBP incubations, although the exact structure of these metab                  Ci/mmol) suspended in 0.2 ml com oil was administered i.p. After 24 h,
                                                                                animals were lightly anesthetized with ether and killed by cervical dislo
olites is not presently known (32).                                             cation. Livers and lungs were removed and stored at -90°C until
   We report here on the DNA-binding levels and adduci profiles
of both 6-NBP and BP in human bronchus and colon expiant                           Incubation of Human Bronchus or Colon Expiants with Either [JHJ-
samples. We have used liver and lung DNA from rats given                        BP or [3H]-6-NBP. Human bronchial and colon tissue were obtained from
injections of either 6-NBP or BP and DNA from Salmonella                        surgical or autopsy specimens and transported       in ice-cold Leibovitz L-
typhimurium incubated with 6-NBP and rat liver S9 for compari                   15 medium. Bronchial expiants were prepared and incubated for 7 days
son. Preliminary metabolism studies from human colon and                        in supplemented CMRL 1066 medium as described previously (35).
bronchus expiants incubated with either 6-NBP or BP are also                    [3H]BP (17.4 Ci/mmol) or [3H]-6-NBP (11.0 Ci/mmol) was added to the
presented.                                                                      medium after 7 days to give a final concentration of 1.5 *JM. Expiants
                                                                                were incubated for a further 24 h, the epithelial layer was removed, and
                                                                                the medium was kept at -90°C until analyzed. Colon expiants were
MATERIALS AND METHODS                                                           incubated for 24 h as reported previously (36) prior to the addition of
                                                                                either [3H]BP or [3H]-6-NBP. Expiants were incubated with carcinogen
   Materials. [G-3H]BP and 14C-labeled calf thymus DNA were obtained            for 24 h and colonie epithelial mucosa were then collected. Culture
from Amersham International PLC, Amersham, United Kingdom; 3-OH-                medium was stored at -90°C until analyzed.
BP was from the National Cancer Institute Chemical Respository, Be-                HPLC Analysis of [3H]BP or [3H]-6-NBP Metabolites in Culture
thesda, MD; BP was from Koch-Light Laboratories, Colnbrook, United              Medium. Media in which expiants had been incubated with either [3H]-
Kingdom; hydroxylapatite (DNA grade) was from Bio-Rad Laboratories,             BP or [3H]-6-NBP were thawed and extracted twice with an equal volume
Watford, United Kingdom; NADP disodium salt, glucose 6-phosphate,               of ethyl acetate:acetone, 2:1. The ethyl acetate layer was then washed
proteinase K, and snake venom phosphodiesterase were from Boehrin-              twice with equal volumes of water before excess anhydrous MgS04 was
ger Corporation (London), Ltd., Lewes, United Kingdom; alkaline phos-           added to remove any water from the extract. The resulting solution was
phatase (type III-S), acid phosphatase (type I), DNase I (type III), and        then filtered and the ethyl acetate was removed under reduced pressure.
nuclease Pi were from Sigma Chemical Company, Poole, Dorset, United             The residues were redissolved in 1 ml benzene and applied to a silica
Kingdom; brain-heart infusion broth was from Difco Laboratories, West           gel column (Kieselgel 60 reinst 70-230 mesh; BDH, Poole, Dorset, United
Molesey, Surrey, United Kingdom; and Sarkosyl NL-30 was obtained                Kingdom). Unreacted [3H]-6-NBP and [3H]BP were eluted with 5 ml
from BDH, Poole, Dorset, United Kingdom, Aroclor-1254 was a gift from           benzene first and the metabolites were then eluted with 8 ml methanol.
ICI Central Toxicology Laboratory, Macclesfield, Cheshire, United King          The methanol was evaporated under reduced pressure and the metab
dom. 7,8-Dtol and BPDEI:dGuo were obtained as a gift from Dr. B.                olites were redissolved in 1 ml methanol and analyzed by HPLC using a
Tiemey, Cancer Research Unit, University of York, York, United Kingdom.         Dupont Zorbax ODS-2 column. The methanohwater gradient used was
All other reagents used were of analytical reagent grade.                       60 to 100% over 50 min. Flow rate was 0.8 ml min fractions were
    Instrumentation. HPLC was carried out using a Pye-Unicam system
                                                                                collected every minute and counted for radioactivity.
comprising two PU4010 pumps, a PU4800 video chromatography control                 Incubation of [3H]-6-NBP with S. typhimurium TA98. S. typhimurium
unit, and a PU4020 UV detector set at 280 nm (Pye-Unicam, Cambridge,
                                                                                TA98 was kindly provided by Professor B. N. Ames, University of
United Kingdom). Samples were injected via a Rheodyne valve (model              California, Berkeley, CA. S. typhimurium TA98 was grown in 200 ml
7125) onto either a Dupont Zorbax ODS-2 column (0.46 x 25 cm) or a
                                                                                brain heart infusion broth overnight. Then 800 ml of the same medium,
Shandon column packed with Partisil-10 ODS-2 (0.6 x 25 cm) (Whatman             previously warmed to 37°C, was added followed by 1.5 mCi of [3H]-6-
Reeve Angel). Flow rates were 0.8 and 2 ml/min, respectively. Radioac           NBP (specific activity, 4 Ci/mmol) dissolved in 1 ml of anhydrous dimethyl
tivity was measured by liquid scintillation counting in a Packard Tri-Carb      sulfoxide. 4.5 mmol of MgCI2,1.5 mmol of NADP, 3 mmol of glucose 6-
300C liquid scintillation counter. Fisofluor scintillant (Fisons Ltd., Lough-   phosphate, 75 mmol of Tris-HCI (pH 7.5), and 20 ml of Aroctor-induced
borough, United Kingdom), 4 ml, was added to 1-ml aqueous samples in            rat liver S9. The mixture was incubated with shaking at 37°C for 4 h.
mini vials. Counts were converted to dpm by automatic external stand
                                                                                  Bacteria were recovered by centrifugation at 10,000 rpm and the cell
ardization using a previously prepared quench curve.                              pellets were stored at -20°C until required.
   Synthesis of [3H]-6-NBP. [3H]BP (specific activity, 21 Ci/mmol) was
                                                                                   Extraction and Purification of DNA from Rat Organs, S. typhimu
nitrated with dinitrogen tetroxide using the method of Radner (33), with
                                                                                rium, and Human Expiant Tissue. DNA was extracted and purified from
modifications. Essentially 12.5 mCi BP were purified on a small alumina         rat liver and lungs using the method of Gupta.3 S. typhimurium DNA was
column using benzene as the eluent. The purified BP was redissolved in
5 ml anhydrous dichloromethane, and 100 ¡A    dinitrogen tetroxide-satu-       extracted and purified from the cell pellets using the method of Stark er
                                                                                al. (37). The final precipitated DNA from these preparations was routinely
rated dichloromethane were added with stirring. The resulting yellow
solution was then stirred for a further 10 min, the dichloromethane was               9R. C. Gupta. Rapid isolation of carcinogen-modified DNA from mammalian
removed under a stream of nitrogen, and the remaining yellow precipitate          tissues, submitted for publication.

                                                  CANCER     RESEARCH       VOL. 45 DECEMBER          1985
                                  BINDING STUDIES AND DNA ADDUCI                 FORMATION         BY BP AND 6-NBP

dried ¡nethanol:acetone (1:1) and acetone before redissolving in 5 mw                                          Tabtet
Bis-Tris (pH 7.1). Mucosae from human bronchus or colon were sus              Binding levels of either [3H]BP or [*H]-6-NBP to purified human bronchus
pended in buffer of the following composition: 0.8 ml 0.24 M NaH2P04.                                        explant DNA
                                                                             For experimental details, see "Materials and Methods." Cases 7-23 inclusive
1% sodium dodecyl sulfate, 10 mw EDTA (pH 6.8), 1 mg proteinase K,
and 200 ng 14C-labeled calf thymus carrier DNA (specific activity, 360     were obtained from the Thorax Clinic, Karolinska Hospital, Sweden. Cases 339-
dpm/200 ng). Samples were incubated for 2.5 h at 37°Cand vigorously       362 inclusive were obtained from the Department of Pathology, University of
                                                                           Maryland, College Park, MD.
shaken every 15 min. After the incubation period, the solutions were
                                                                                                                   pmol compound/10 mg DNA
saturated with urea and ultrasonicated for 30 s in an A350G Ultrascni-
cator fitted with a 3-mm probe (Ultrasonics Ltd.). Samples were then                        Case7810111213142123339341344A346346B3473626-NBP19.765.989.065.967.4
applied to a hydroxylapatite column made up from 2 g hydroxylapatite
suspended in 0.24 M NaH2PO4:1% sodium dodecyl sulfate: 10 ITIM
EDTA:8 M urea (pH 6.8). RNA and protein were first eluted with 0.24 M
NaH2PO4:8 M urea (pH 6.8) and DNA was then eluted with 0.48 M
NaH2PO4 (pH 6.8). The isolated DNA fractions were dialyzed against
water for 24 h and then adjusted to 0.1 M NaCI before precipitating the
DNA with excess ethanol overnight at -20°C. The resulting precipitates
were redissolved in 3 ml 5 mw Bis-Tris (pH 7.1) and the DNA concentra
tion was determined by measuring the UV absorbance at 260 nm.
Radioactivity was measured by liquid scintillation counting.
   Enzymic Digestion and HPLC Analysis of DNA from Rat Liver and
Lung, Human Expiants, and S. typhimurium. Liver and lung DNA from
the rat, human expiant, and S. typhimurium TA98 DNA dissolved in 5
m«Bis-Tris (pH 7.1) was enzymically hydrolyzed with DNase I, alkaline
phosphatase. acid phosphatase, nuclease P,, and snake venom phos-                           Mean ±SD                 22.4
                                                                                                                50.9 ±               42.0 ±15.5
phodiesterase following the procedure of Martin e?al. (30). Hydrolysates
were extracted twice with equal volumes of water-saturated butano!.
The butanol layers were separated and evaporated under reduced
pressure. Residues were then redissolved in 1 ml 30% methanol:water                                               Table2
and analyzed on HPLC using an analytical Shandon column packed with            Binding levels of either [3H]BP or [3H]-6-NBP to human colon explant DNA
Partisil-10 ODS-2. The methanol:water     gradient was 30% metha-             For experimental details, see "Materials and Methods." Cases were obtained
nol:water for 10 min, followed by 30 to 70% methanol:water over 10         from Department of Pathology, Universityof Maryland.
min, followed by 70 to 100% methanol:waterover 5 min. The 6-NBP and
                                                                                                                   pmol compound/10 mg DNA
B P markers used were not separable on this system. The flow rate was
                                                                                               Case               6-NBP                    BP
2 ml/min and the eluent was monitored at 280 nm. Fractions were
collected every 0.5 min and counted for radioactivity.                                      257                                           77.0
                                                                                            269                    27.0
                                                                                            270A                   33.0                   45.0
                                                                                            289A                   40.0                   59.7
RESULTS                                                                                     289B                   40.0                   98.7
                                                                                            289D                                          51.9
  Binding of [3H]-6-NBP or [3H]BP to Human Bronchus and
                                                                                            Mean ±SD            35.0 ±6.2           66.5 ±21.6
Human Colon Expiant Samples. Incubation of human bronchus
or colon expiants with either [3H]BP or [3H]-6-NBP for 24 h
resulted in the conversion of these compounds to DMA-bound
species (Tables 1 and 2). Although the number of samples is
limited, there appeared to be little difference in the levels of                                               Tabled
                                                                               Percentageof BP and 6-NBPmetabolized by human bronchus and colon expiant
binding between the two compounds in either bronchial or colon                             samples to aqueousand organic-soluble metabolites
specimens.                                                                   All cases were obtained from Department of Pathology, Universityof Maryland,
   Analysis of [3H]BP or [3H]-6-NBP Metabolites in Culture                 except Cases 21 and 23 which came from the Thorax Clinic, Karolinska Hospital,
Medium from Bronchus and Colon Incubations. Limited anal                   Sweden.
yses of expiant culture media after incubation with [3H]BP or                                                      % of
[3H]-6-NBP indicated that both compounds were converted to
both water- and organic-soluble metabolites (Table 3). Total
percentage of metabolism for BP was 11-23% in the colon and
8-59% in the bronchus. The equivalent figures for 6-NBP were
36-50% in the colon and 18-41% in the bronchus. The organic-
soluble metabolites from colon and bronchus incubations were
analyzed by reverse-phase HPLC. Using authentic marker com
pounds, 7,8-diol and 3-OH-BP were identified as two of the
metabolites present in both colon and bronchus incubated with
[3H]BP (data not shown). Other metabolites present were not
identified in this study. Analysis of the 6-NBP organic-soluble
metabolites revealed a less complex pattern. One major metab
olite of 6-NBP was detected in both colon and bronchus (data                      3 Percentage orgamc-extractablemetabolites excluding residual starting mate
not shown) that cochromatographed with the authentic 3-OH-                     rial.

                                               CANCER     RESEARCH      VOL. 45 DECEMBER              1985
                                     BINDING STUDIES AND DNA ADDUCI                  FORMATION       BY BP AND 6-NBP

BP marker. Preliminary studies indicate that this metabolite peak
also cochromatographs with the major metabolite formed when
rat microsomes are incubated with 6-NBP (data not shown),
which has previously been identified as 3-OH-6-NBP (38).
   Analysis of Enzymically Hydrolyzed DNA from Human Bron
chus Incubated with [3H]BP or [3H]-6-NBP. HPLC analysis of
enzymically hydrolyzed DNA from human bronchus expiants
previously incubated with either [3H]BP or [3H]-6-NBP revealed
one and two major adducts, respectively (Chart 1). Both 6-
NBP:DNA adducts eluted earlier than did the major BP:DNA
   Analysis of Enzymically Hydrolyzed DNA from S. typhimu-
rium Incubated with [3H]-6-NBP. Incubation of S. typhimurium
TA98 with [3H]-6-NBP and Aroclor-induced rat liver S9 resulted
in the generation of a possible three DNA adducts (Chart 2). The
second adduct had the same retention time as did the major
adduct isolated from human bronchus DNA incubated with 6-
                                                                                                k                                  i

                                                                                      Chart 2. Reverse-phase HPLC profile of enzymic hydrolysates of bacterial DNA
NBP and with the major liver and lung DNA adduct from rats                         obtained after incubation of [3H]-6-NBP rat liver S9 and S. typhimurium TA98.
given injections of 6-NBP.                                                         HPLC conditions are as described in "Materials and Methods." Marker compounds
                                                                                   were BPDEIidGuo (/) and 6-NBP (//).
   Analysis of Enzymically Hydrolyzed DNA from Liver and
Lungs of Rats Given i.p. injections of [3H]BP and [3H]-6-NBP.
                                                                                   [3H]BP revealed three adducts in liver DNA (Chart 3A) and two
HPLC analysis of enzymically hydrolyzed DNA from rats given
                                                                                   adducts in lung DNA (Chart 44). In both organs, peak II cochro-
                                                                                   matographed with the authentic BPDEhdGuo marker but the
                                                                                   major DNA adduct eluted 2 min earlier. Only one adduct was
                                                                                   detected in the enzymic hydrolysate of DNA from the liver and
                                                                                   lungs of rats given injections of [3H]-6-NBP (Charts 3ßand 4B).


                                                                                      PAH are widely distributed environmental pollutants that have
                                                                                   been implicated in the etiology of human cancer (10). However,
                                                                                   mutagenic activity of air particulates collected from urban loca
                                                                                   tions cannot all be attributed to PAH. Interest therefore has
                                                                                   recently been focused on the nitrated PAH due to the finding of
                                                                                   their possible facile formation in the environment (39) and their
                                                                                   potent direct-acting mutagenicity in bacteria (24). Little data exist
        1                                                                          concerning either the metabolism or DNA binding of this group
                                                                                   of compounds. The most intensively investigated to date is 1-
                                                                                   nitropyrene, a carcinogen in experimental animals (40) which has
                                                                                   been shown to form an A/-(deoxyguanosin-8-yl)-1-aminopyrene
                                                                                   adduct in S. typhimurium TA1538 (28) and in vivo in rats given
                                                                                   1-nitropyrene (29), indicating the importance of the nitro group
                                                                                   in the activation of this compound. Conversely 6-NBP, which is
                                                                                   not a direct-acting mutagen in bacteria, appears to be metabo
                                                                                   lized to primarily phenolic metabolites when incubated with rat
                                                                                   liver microsomes (31, 32) and dihydrodiols in whole cell systems
                                                                                   such as hamster embryo fibroblasts (32). While we have not
                                                                                   studied the metabolism of 6-NBP in depth, it does appear that
                                                                                   human bronchus and colon expiants can convert 6-NBP to a
                                                                                   metabolite that cochromatographs with the only product formed
                                                                                   when rat microsomes are incubated with 6-NBP. This has been
                                                                                   identified by nuclear magnetic resonance and mass spectra as
                                                                                   3-OH-6-NBP (38) thus indicating that ring oxidation may also be
                                                                                   a major metabolic pathway in human tissue. However, because
                                                                                   no other metabolites were detected, there was no evidence for
   Chart 1. Reverse-phase HPLC profiles of enzymic hydrolysates of DNA from        the formation of dihydrodiols. The media from BP incubations
human bronchus expiants incubated with either [3H]BP (A) or [3H]-6-NBP (B). HPLC
conditions are described in "Materials and Methods." Marker compounds used in
                                                                                   with colon and bronchus included both 3-OH-BP and 7,8-diol,
A were BPDEhdGuo (/) and BP (II) and in 8 they were BPDEhdGuo (I) and 6-NBP        although no attempt was made to separate these from the
(II).                                                                              closely eluting 4,5-diol and 9-OH-BP, which may also be present.

                                                    CANCER      RESEARCH       VOL. 45 DECEMBER          1985
                                     BINDING STUDIES AND DNA ADDUCT FORMATION                        BY BP AND 6-NBP

                                                                                   O    BOO.

                                                                                        6 O O.


                         2O        30        40           50       60
                                                   FRACTION NUMBER

                                                                                                                         rTrf   flrrim
               li                                                                     Chart 4. Reverse-phase HPLC profiles of enzymic hydrolysates of lung DNA
                                                                                   obtained after administration of [3H]BP (A) and [3H]-6-NBP (8) to rats. HPLC
                                                  FRACTION   NUMBER
                                                                                   conditions are described in 'Materials and Methods." Marker compounds used in
   Charts. Reverse-phase HPLC profiles of enzymic hydrolysates of liver DNA        A were BPDEhdGuo (/) and BP (//), and in B they were BPDEhdGuo (/) and 6-NBP
obtained after administration of [3H]BP (A) and [3H]-6-NBP (8) to rats. HPLC
conditions are described in "Materials and Methods." Marker compounds used in
A were BPDEhdGuo (/) and BP (II), and in 8 they were BPDEI:dGuo (I) and 6-NBP
                                                                                   (41), esophagus (46), and alveolar macrophages, respectively
                                                                                   (43). Another reason may be the different anatomical origin of
7,8-Diol has been identified previously as the major metabolite                    the expiants. It is known, for instance, that binding tends to be
formed when BP was incubated with either colon or bronchus                         higher in the ascending and traverse colon than in other sections
and is most probably the precursor to the active DNA-binding                       of the colon (36). Although the methods used here are similar to
species (36, 41-43). Total metabolism of 6-NBP was similar in                      those previously reported, slight changes in technique may also
colon and bronchus expiant incubations; however, BP metabo                         account for these differences, as well as the fact that the number
lism was generally higher in the bronchus, as has been reported                    of cases studied here is limited. HPLC analysis of enzymic DNA
previously (36).                                                                   digests revealed a number of apparent adducts formed by 6-
   Previous reports showed that a greater proportion of these                      NBP and BP in bacterial, rat, and human DNA. Although many
metabolites were water soluble. They were thought to be sulfate                    attempts were made to find a solvent gradient that would satis
esters and glutathione conjugates of diols, tetrols, and possibly                  factorily resolve the adducts, the system presented here was
their epoxide intermediates which have been found to be good                       found to be the most suitable. The 6-NBP and BP markers used
substrates for UDP-glucuronyltransferase (44). We found that 6-                    in each instance were not separable under these conditions, a
NBP bound to the DNA of human colon and bronchus expiant                           fact that we used extensively when interpreting the HPLC traces.
samples at levels similar to that of BP. In both cases, the range                  HPLC analysis of enzymic DNA digests of human bronchus after
between individuals was within one order of magnitude. Values                      exposure to 6-NBP indicated a possible two adducts. Insufficient
reported here are somewhat higher than those reported previ                        DNA was recovered from human colon for adduci analysis. One
ously (35, 36). These differences may be accounted for by                          of the major 6-NBP:DNA adducts isolated from human bronchus
interindividual variations. For example, previous investigators                    expiants had the same retention time as did the major 6-
have recorded a 75-, 100-, 99-, and 9-fold variation between                       NBP:DNA adduct isolated from liver and lung of rats given 6-
individuals for BP binding to DNA of the bronchus (45), colon                      NBP and the major DNA adducts isolated from S. typhimurium

                                                     CANCER      RESEARCH   VOL. 45 DECEMBER            1985
                                        BINDING STUDIES AND DNA ADDUCI                    FORMATION         BY BP AND 6-NBP

TA98 incubated with 6-NBP and liver S9. This would appear to                             9. Deckers-Schmelzler, B., Klaus, R., and Kahl, G. F. Binding of benzo(a)pyrene
                                                                                            metabolites to cellular DNA in perfused rat lung. Naunyn-Schmiedebergs Arch.
indicate that 6-NBP may be metabolized by a similar pathway in                              Pharmakol., 303: 303-307,1978.
humans, rats, and bacteria. We further investigated BP:DNA                              10. Kahl, G. F., Klaus, E., Legraverard, C., Nebert, D. W., and Pelkonen, O.
                                                                                            Formation of benzo(a)pyrene metabolite-nucleoside adducts in isolated per
adduct formation in human bronchus expiants and rat liver and                               fused rat and mouse liver and mouse lung slices. Biochem. Pharmacd., 28:
lung in order to make comparisons with 6-NBP:DNA adduct                                     1051-1056,1979.
formation in the same systems. Only one adduct was detected                             11. Vahakangas, K., Nebert, D. W., and Pelkonen, O. The DNA binding of benzo-
                                                                                            (a)pyrene metabolites catalysed by rat lung microsomes in vitro and in isolated
in the enzymic hydrolysates of DNA from human bronchus                                      perfused rat lung. Chem.-Biol. Interact., 24:167-176,1979.
expiants previously incubated with BP. This cochromatographed                           12. Moore, B. P., and Cohen, G. M. Metabolism of benzo(a)pyrene and its major
                                                                                            metabolites to ethyl acetate-soluble and water-soluble metabolites by cultured
with BPDEI:dGuo and eluted later than did the adducts formed                                rodent trachea. Cancer Res., 38: 3066-3075,1978.
from the 6-NBP incubations. The isolation of this adduct is                             13. Phillips, D. H., Grover, P. L., and Sims, P. The covalent binding of polycydic
consistent with the data reported previously (6,7). Although this                           hydrocarbons to DNA in the skin of mice of different strains. Int. J. Cancer,
                                                                                            22: 487-494,1978.
adduct was also isolated from DNA of liver and lungs from rats                          14. Adriaenssens, P. I., White, C. M., and Anderson, M. W. Dose-response
given [3H]BP, it was not the major adduct, which eluted some 2                              relationships for the binding of benzo(a)pyrene metabolites to DNA and protein
                                                                                            in lung, liver and forestomach of control and butylated hydroxyanisole-treated
min earlier on HPLC. These results are in general agreement
                                                                                            mice. Cancer Res., 43: 3712-3719,1983.
with those of Boroujerdi ef al. (17) who showed that the major                          15. Kulkami, M. S., and Anderson, M. W. Persistence of benzo(a)pyrene metabo
adduct formed in vivo in the rat following i.v. administration of                           lite: DNA adducts in lung and liver of mice. Cancer Res., 44: 97-101,1984.
BP was not the same as that reported previously for certain                             16. Wilson, A. G. E., Kung, H. C., Boroujerdi, N., and Anderson, M. W. Inhibition
                                                                                            in vivo of the formation of adducts between metabolites of benzo(a)pyrene
strains of mice (14-16) and in various in vitro systems (2-13). It                          and DNA by aryl hydrocarbon hydroxylase inducers. Cancer Res., 41: 3453-
was postulated that the major adduct formed in the rat may be                               3460,1981.
the result of the further metabolism of 9-OH-BP, possibly to 9-                         17. Boroujerdi, N., Kung, H. C., Wilson, A. G. E., and Anderson, M. W. Metabolism
                                                                                            and DNA binding of benzo(a)pyrene in vivo in the rat. Cancer Res., 41: 951-
OH-BP-4,5-epoxide (17). Alternatively the adduct may be a deox-                             957, 1981.
ycytosine product (14-16). It is interesting to note that the major                     18. Alexandrov, K., and Thompson, M. H. Influence of ¡nducersand inhibitions of
                                                                                            mixed-function oxidases on benzo(a)pyrene binding to the DNA of rat liver
6-NBP:DNA adduct in rat liver and lung, human bronchus ex-
                                                                                            nuclei. Cancer Res., 37:1443-1449,1977.
plant, and one of the adducts isolated from S. typhimurium TA98                         19. King, H. W. S., Thompson, M. H., and Brookes, P. The rote of 9-hydroxy-
elute at the same retention time as does the major BP:DNA                                    benzo(a)pyrene in the microsome mediated binding of benzo(a)pyrene to DNA.
                                                                                             Int. J. Cancer, 18: 339-344, 1976.
adduct in rat liver and lung DNA. Because BP and 6-NBP markers
                                                                                        20. Jemstrom, B., Orrenius, S., Undeman, O., Graslund, A., and Ehrenberg, A.
were not separable under the HPLC conditions used, it is tempt                               Fluorescence study of DNA-binding metabolites of benzo(a)pyrene formed in
                                                                                             hepatocytes isolated from 3-methylcholanthrene-treated        rats. Cancer Res.,
ing to suggest that the adducts they form may be structurally
                                                                                            38: 3600-3607,1978.
similar arising through a diol-epoxide intermediary. However,
                                                                                        21. Gibson, T. Nitroderivatives of polynuclear aromatic hydrocarbons in airborne
while one might speculate on the structures of the 6-NBP adduct,                             and source paniculate. Atmos. Environ., 76: 2037-2040,1982.
we do not have as yet definitive evidence for its structure. In                         22. Schuetzle, D., Ritey, T. L., Prater, T. J., Harvey, T. M., and Hunt, D. F. Analysis
                                                                                             of nitrated polycydic aromatic hydrocarbons in diesel particulates. Anal. Chem.,
conclusion, the finding that 6-NBP binds to human explant DNA                                54:265-271,1982.
lends credence to the idea that nitro-PAH could pose a carcino                          23. Schuetzle, D., Ritey, T. L., Prater, T. J., and Salmeen, I. The identification of
                                                                                             mutagenic chemical spedes in air paniculate samples. In: J. Albaiges (ed.),
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                                                                                             Analytical Techniques in Environmental Chemistry, Vol., 2, Oxford, United
culture, some are carcinogenic in animals, and many occur widely                             Kingdom pp. 259-280. Pergamon Press, Ltd., 1982.
in the environment. For this reason, the sources of these com                           24. Mermelstein, R., Kiriazides, D. K., Butler, M., McCoy, E. C., and Rosenkranz,
                                                                                             H. S. The extraordinary mutagenicity of nitropyrenes in bacteria. Mutât.Res.,
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                                                     CANCER       RESEARCH       VOL. 45 DECEMBER             1985

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