Carcmogewsis vol.14 no.10 pp.2137-2141, 1993
Promotion of preneoplastic lesions and induction of CYP2B by
unleaded gasoline vapor in female B6C3F1 mouse liver
Andrew M.Standeven and Thomas L.Goldsworthy1
Department of Experimental Pathology and Toxicology, Chemical Industry
Institute of Toxicology, Research Triangle Park, NC 27709, USA
'To whom correspondence should be addressed
Introduction
The US Environmental Protection Agency has estimated that
>3.6 billion gallons of unleaded gasoline (UG*) are released
into the air as vapors annually, with - 4 0 % of this occurring
at retail service stations (1). With over 100 million people
pumping UG at self-service stations (2), the potential for human
exposure to UG vapors is great. A two-year cancer bioassay
conducted in the late 1970s demonstrated that inhalation exposure
of rodents to a reference blend of UG designated 'PS-6' caused
an increase in kidney tumors in male rats and liver tumors in
female mice (3). While the kidney tumors have been attributed
to interaction of UG components with a male rat-specific renal
•Abbreviations: UG, unleaded gasoline; DEN, JV-nitrosodiethylamine; PROD,
pentoxyresorufm-0-dealkylase; BrdU, 5-bromo-2'-deoxyuridine.
Materials and methods
Chemicals
PS-6 blend and API 9 1 - 1 blend UG were generously donated by the American
Petroleum Institute (Washington, DC). The PS-6 blend UG, the composition of
which has been published (3), was from the same lot used in the cancer bioassay.
As compared to PS-6, API 9 1 - 1 blend UG contains a greater percentage of
aromatics(33.2% versus 26.1%) and olefins (12.596 versus 8.4%) and a lower
percentage of saturated hydrocarbons (53.1 % versus 65.5%). Neither blend
contains significant levels of oxygenates (3,14,15). Unless otherwise specified,
all other chemicals were obtained from Sigma Chemical Co. (St Louis, MO).
Animals
All experiments were conducted under NIH guidelines for the care and use of
laboratory animals and were approved by the CUT Institutional Animal Care and
Use Committee. For the gavage experiment, 'virus-free' female B6C3F1 mice
were obtained from Charles River Breeding Labs (Raleigh, NC) and acclimated
for 10 days. Mice were housed individually in polystyrene cages on alpha cellulose
bedding in a temperature- and humidity-controlled room. Mice were kept on a
12 h light/dark cycle, with the light period extending from 6 a.m. to 6 p.m. Food
(NIH-07 Open formula diet; Ziegler Bros., Gardners, PA) and filter-purified tap
water were provided ad libitum. For the inhalation experiment, 'virus-free' male
C3H/HeNCrlBR mice and female C57BL/6NCrlBR mice were obtained from
Charles River (Raleigh, N Q and acclimated to the conditions described above.
The mice were then bred and the resulting B6C3F1 offspring were treated as
described below.
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An initiation-promotion protocol was used to test the
hypothesis that unleaded gasoline (UG) vapor acts as a liver
tumor promoter in female mice under exposure conditions
in which UG was hepatocarcinogenk in a cancer bioassay.
Twelve day old female B6C3F1 mice were injected with
A'-nitrosodiethylamine (DEN, 5 mg/kg, i.p.) or vehicle.
Starting at 5 - 7 weeks of age, mice were exposed by inhalation
6 h/day, 5 days/week for 13 weeks to 0 or 2039 p.p.m. of
PS-6 blend UG, the same gasoline blend used in the cancer
bioassay. Putative preneoplastic lesions in liver, characterized
mainly as basophilic foci in H&E-stained liver sections, were
found exclusively in mice treated with DEN. While similar
numbers of altered hepatic foci were found in DEN-initiated
mice treated with 0 or 2039 p.p.m. UG, UG treatment
significantly increased both the mean volume (3.2-fold) and
the volume fraction (3.6-fold) of the foci. To determine if UG
induced CYP2B, a subfamily of cvtochrome P450 commonly
induced by liver tumor promoters in rodents,
pentoxyresonifin-0-dealkylase (PROD) activity was assayed
in hepatic microsomes derived from the above livers. UG
vapor increased hepatic PROD activity ~ 8-fold, while
increasing cytochrome P450 content only -30%. To ascertain
if a more recent blend of UG, API 9 1 - 1 , would have similar
biological effects as PS-6, female B6C3F1 mice were gavaged
for 3 days with corn oil or 1800 mg/kg/day PS-6 or API 9 1 - 1
blend UG. PS-6 and API 9 1 - 1 blend UG induced similar
increases in relative liver weight (-25%), PROD activity
(~ 9-fold) and hepatocyte labeling index (—8-fold) relative
to controls. These data demonstrate that PS-6 blend UG vapor
promotes preneoplastic lesions and induces CYP2B in female
mouse liver under exposure conditions in which it causes liver
tumors, and suggest that a more recent blend of UG may have
similar effects.
protein (4), and thus are generally regarded as inappropriate for
human risk assessment (5), the relevance of the mouse liver
tumors induced by UG for human risk assessment remains
unclear.
UG has produced negative or weakly positive results in a
number of short-term assays for mutagenicity and genotoxicity
(6—8). In a 90 day inhalation study, exposure of mice and rats
to UG vapors caused hepatomegaly and a transient increase in
hepatocyte proliferation in the absence of hepatotoxicity (9). Such
effects are characteristic of a class of non-genotoxic carcinogens
called mitogens, many of which act as promoters in two-stage
models of cardnogenesis (10). Moreover, Brady et al. (11) found
that i.p. injection of rats with UG induced hepatic CYP2B1, a
P450 isoform induced as part of a pleiotropic response common
to a number of rodent liver tumor promoters (12,13). However,
the ability of UG to function as a liver tumor promoter has not
been directly tested, and it is not known if UG induces CYP2B
under the conditions of the cancer bioassay.
In the present study, an initiation—promotion protocol, with
altered hepatic foci as the endpoint, was used to test the hypothesis
that PS-6 blend UG acts as a liver tumor promoter in female
mice under conditions in which it is hepatocarcinogenic. Hepatic
CYP2B induction was also examined. In addition, since the
composition of UG has changed since the cancer bioassay was
conducted, the short-term effects of PS-6 and a more recent blend
of UG, API 91 - 1 , on various endpoints potentially relevant to
tumor promotion were compared. Our data indicated that PS-6
blend UG promoted growth of hepatic preneoplastic lesions and
induced CYP2B under bioassay exposure conditions. Moreover,
PS-6 and API 9 1 - 1 blend UG had similar acute effects in mouse
liver, suggesting that API 91 — 1 may share the promotional
activity of PS-6.
A.M.Standeven and T.L.Goldsworthy
Necropsy
Approximately 20 h after the last inhalation exposure, mice were weighed,
anesthetized with isoflurane and exsanguinated, livers were removed, weighed
and examined for the presence of gross lesions. Sections of the left, median right
and right anterior lobes were fixed in 10% buffered formalin. The balance of
the liver was minced, rinsed with ice-cold isotonic KCl-Tris (0.154 M KC1,
0.050 M Tris, pH 7.4), placed on ice, and used to prepare microsomes as described
below. Between 24 and 48 h after necropsy, the formalin was replaced by 70%
ethanol. Tissues were embedded in paraffin, sectioned at 5 pm, stained with H&E,
and examined microscopically.
Quantiuakm of hepatic preneoplastic lesions
The total area of liver at each sample site occupied on an H&E-stained section
from the inhalation experiment was determined with an Image-1 image processing
system (Universal imaging Corp., West Chester, PA). Sections were examined
for the presence of altered hepatic foci a 10 cells in size with the experimenter
band to the treatment group, and foci were rlmmfind according to histopathological
phenotype using standard criteria (16). The area of each focus was recorded and
used to calculate the number and volume of foci according to the stereological
method of Pugh et al. (17) using a focal profile cutoff with radius = 65 fim.
Microsome preparation
Washed microsomes were prepared by modifications of existing procedures
(18,19). Briefly, minced liver pooled from two or three mice per group was
homogenized in 3 vol of ice-cold isotonic KCl-Tris with a 5 s burst of a
Tissumizer homogenizer. The homogenate was centrifuged at 10 000 g for 20
min at 4°C. The supernatant was diluted with 3 vol of isotonic KCl-Tris and
centrifuged at 100 000 g for 60 min at 4°C. The pellet wasresuspendedmanually
in 0.05 M Tris/0.25 M sucrose/1.0 mM EDTA (pH 7.4) and centrifuged at
100 000 g for 60 min at 4°C. The microsomal pellet was resuspended manually
with a glass—glass homogenizer in 0.1 M sodium phosphate/0.25 M sucrose
(pH 7.4) and frozen at - 8 0 ° C until assay.
Microsomal assays
Peatoxyresorufin-0-dealkylase (PROD) activity was assayed essentially as
described by Lubet et al (20).
The cytochrome P450 content was determined from the dithionite difference
spectrum of CO-treated microsomes using an extinction coefficient of 104 mM" 1
cm" 1 (21).
Microsomal protein was assayed with Coomassie-Plus Protein Assay Reagent
(Pierce, Rockford, IL) using bovine serum albumin (Pierce) as a standard.
PS-6 and API 91—1 comparison experiments
Experiment 1. Onday 1 ( — 3 p.m.), two groupsof eight female B6C3F1 mice,
8 — 10 weeks old, were implanted s.c. with osmotic pumps (Alzet model 2001,
1 /J/h; Aba Corp., Palo Alto, CA) containing 16 mg/ml 5-bromo-2'-deoxyuridine
(BrdU) dissolved in PBS (Gibco-BRL, Bethesda, MD). The pH of the BrdU
solution was adjusted to 7.2 ± 0.2 with sodium hydroxide. On days 2 - 4 (-9.30
a.m), mice were treated by i.g. intubation with com oil or 1800 mg/kg of PS-6
blend UG in com oil in a volume of 5.0 ml/kg. On day 5 ( — 9.30 a.m.), mice
were killed and necropsied as described above, except that blood was obtained
by cardiac puncture and used to prepare serum. Sorbitol dehydrogenase activity
in serum was assayed mmnvliatfly using a commercial kit (Sigma no. 50-UV)
and a Roche Cobas Farra U clinical analyzer. Microsomes were prepared and
PROD activity was measured as described above.
Experiment!. Experiment 2 was identical to experiment 1, except that mice were
treated with either com oil or 1800 mg/kg/day of API 91 - 1 blend UG.
2138
Immunohistochemistry
BrdU incorporation in imstained liver sections was determined by modifications
of the procedure of FJdridge et al. (22). Briefly, deparaffinized tissue sections
were incubated in 3% hydrogen peroxide for 10 min. The tissue was digested
by incubation in 0.1% pepsin dissolved in 0.01 N HC1 (pH 2.25) for 10 min,
followed by incubation in 2 N HC1 at 37°C for 30 min. Non-specific binding
sites were blocked by incubation with normal horse serum for 10 min, and a
1:50 dilution of primary antibody (anti-mouse BrdU, Becton Dickinson, San Jose,
CA) in 0.05% bovine serum albumin was applied for 1 h. A 1:200 dilution of
anti-mouse IgG antibody (Vector Lab, Burlingame, CA), followed by alkaline
phosphatase-conjugaled streptavidin (Vector Lab), were then applied for 30 min
each. After application of phosphatase enhancer (Biomeda Corp., Foster City,
CA) for 3 min, tissue sections were incubated with Fast Red chromagen (Biomeda
Corp.) for 8 min. Nuclei were counterstained with hematoxylin (Biomeda Corp).
Determination of labeling index
BrdU-stained liver sections were examined by light microscopy with the
experimenter blind to the treatment group. Computer-generated random fields
were identified to score nuclei for BrdU incorporation. At least 2000 hepatocellular
nuclei in the left lobe were scored. Hepatocyte labeling index was calculated by
dividing the number of labeled cells by the total number of cells scored, and
multiplying by 100.
Statistics
Non-quanta] data are presented as the mean ± SD. Means in Table I and Figure 1
were compared by one-way analysis of variance followed by Schefte's test if
significant differences were found. The focal parameters for the DEN/control
and DEN/UG groups were compared with an unpaired, two-tailed r-test, and the
incidence of gross lesions in these mice was compared by chi-square analysis
(Table II). Parameters of UG and com oil-treated mice from the same experiment
(Table DT) were compared using an unpaired, two-tailed /-test. Differences were
considered significant at P < 0.05.
Results
Initiation-promotion experiment
Promotion treatment was not begun until the mice were 5 - 7
weeks old, since that is the age at which UG exposures were
begun in the cancer bioassay of UG (3). Treatment with DEN,
UG or both did not significantly affect body weight at any time
point during the 13 weeks of inhalation exposure (Figure 1). One
saline/UG-treated mouse died as a result of a cage accident. There
were no remarkable clinical observations except hair loss on the
legs and/or abdomen of a few mice beginning on week 8. This
hair loss occurred in mice from all groups and was probably
caused by rubbing against the metal caging.
34
32"
Silmc/Control
SaHne/UG
DEN/Comrol
DEN/UG
24-
22-
20
1
2
3
4
5
6
7
8
9
10
111213
Weeki of Promotion
Fig. 1. Weekly body weights for mice in different treatment groups. Values
shown are the mean of seven or eight mice per group. Error bars have been
omitted for clarity; however, the coefficient of variation for the body
weights did not exceed 11 % for any group at any time.
Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on September 16, 2016
Initiation—promotion experiment
Animal treatments
At exactly 12 days of age, female B6C3F1 mice were injected i.p. with either
5.0mg/kgAr-nitrosodiethylamine(DEN)in0.9% NaClorO.9% NaQalone (7.1
ml/kg). The mice were weaned at 4—6 weeks of age and housed individually
as described above.
At 5 - 7 weeks of age, the B6C3F1 mice from the DEN initiation and NaCl
control groups were separately randomized by weight, assigned to one of two
groups (n = 8), and transferred to individual hanging stainless sted cages contained
in a 1 m3 whole-body inhalation chamber. The mice were exposed to 0 or 2056
p.p.m. (target concentration) of wholly vaporized PS-6 blend UG for 6 h/day,
5 days/week, for 13 weeks. Exposures were routinely conducted from —8.00
a.m. to 2.00 p.m. on weekdays, including holidays. The chamber design, exposure
generation system and monitoring system were exactly as described previously
(9), with chamber concentrations of UG determined hourly. Average dairy chamber
concentrations of UG ranged from 1678 to 2193 p.p.m., with a mean and standard
deviation of 2039 ± 64 p.p.m. (99.2% of target level). Filter-purified tap water
was available ad libitum, whereas food (pelletized NIH-07 Open Formula diet)
was only available during non-exposure periods. Clinical observations and body
weights were recorded weekly.
Promotion of preneoplastfc lesions by
Hepatic non-neoplastic findings
UG treatment alone increased relative liver weight by 11 %, which
is in good agreement with the 10% increase in relative liver
weight observed after exposure of female mice to 2056 p.p.m.
UG for 13 weeks in the cancer bioassay of UG (23). The
combination of DEN and UG increased relative liver weight by
20%, and this increase was significantly greater than that caused
by UG alone (Table I). Inhalation exposure to UG caused a 7to 8-fold increase in hepatic PROD activity and a 29-35%
increase in microsomal cytochrome P450 content with or without
prior DEN treatment (Table I). Midlobular and/or centrilobular
hypertrophy was observed in all mice treated with UG but in
none of the mice exposed to control air, regardless of prior DEN
treatment.
As indicated in Table II, UG treatment of DEN-initiated mice
did not significantly change the total number of foci relative to
DEN controls. However, UG treatment of DEN-initiated mice
resulted in substantial increases in the mean volume of the hepatic
foci (3.2-fold) and in the volume fraction of liver occupied by
foci (3.6-fold) relative to the DEN/control group (Table II). While
the pale white masses observed grossly were generally too small
to be sampled individually, those that were examined had the
appearance of large basophilic foci. Thus, the increased mean
volume and volume fraction of the foci in the DEN/UG group
corroborated the gross finding of a higher incidence of masses
in this group.
PS-6 and API 91—1 comparison experiments
To extend the above findings obtained with PS-6 blend UG to
a more recent blend of UG, API 91 — 1, we sought an acute
exposure regimen that would mimic the hepatic effects of
subchronic inhalation exposure to UG. A preliminary
dose-response study indicated that i.g. treatment of female
B6C3F1 mice with 1800 mg/kg/day of PS-6 blend UG for 3 days
increased relative liver weight, whereas 200 or 600 mg/kg/day
of PS-6 did not increase liver weight (data not shown). Since
hepatomegaly was also observed in the inhalation experiment,
a dose of 1800 mg/kg/day was used to compare PS-6 and API
91 - 1 . As shown in Table m, neither blend of UG affected final
body weight or caused hepatotoxicity as judged by sorbitol
dehydrogenase activity in serum. Histopathological examination
of H&E-stained liver sections revealed mild centrilobular
hypertrophy, but no hepatic necrosis in UG-treated mice. Gavage
Table I. Final body weight, relative liver weight, hepatic cytochrome P450 levels, and hepatic PROD activity in female B6C3F1 mice after treatment with
DEN and/or UG
Treatment1
Saline/control
Saline/UG
DEN/control
DEN/UG
Body wt
(g)
8
7
8
8
30.6
30.6
29.6
29.9
±
±
±
±
Liver wt
((% body wt)
2.6
2.1
3.0
1.1
5.33
5.92
5.37
6.46
±
±
±
±
0.36
0.23d
0.25
0.42^' f
Hepatic
PROD
Hepatic
cytochrome
P450b
1.33
1.80
1.30
1.71
±
±
±
±
18.6
142.4
20.2
150.5
0.08
0.02*
0.09
0.07*
± 2.9
± 21.l d
± 3.7
±
*Mice were injected i.p. with DEN (5 mg/kg) or saline at 12 days of age. Beginning at 5—7 weeks of age, mice were exposed to 0 or 2039 p.p.m. UG
vapor 6 h/day, 5 days/week for 13 weeks.
b
nmol/mg microsomal protein (n = 3, pooled from two or three mice per sample),
'pmol resorufin/min/mg microsomal protein (n = 3, pooled from two or three mice per sample).
Significantly different from saline/control group.
'Significantly different from DEN/control group.
Significantly different from saline/UG group.
Table n . Incidence of gross hepatic masses and parameters of bepatocellular foci in female B6C3F1 mice treated with DEN and/or UG vapor
Treatment1
Saline/control
Saline/UG
DEN/control
DEN/UG
8
7
8
8
Incidence
Hepatic foci
gross
masses
Density
(no./liver)b
Mean volume
(mm3)
Volume fraction
0/8
0 ± 0
0 ± 0
357 ± 240
403 ± 147
0 ± 0
0 ± 0
19.5 ± 7.9 (I.Of
61.7 ± 36.8"(3.2)
0 ±
0 ±
0.39
1.39
on
11%
7/8d
0
0
± 0.23 (1.0)
± 0.97d(3.6)
•Mice were injected i.p. with DEN (5 mg/kg) or saline at 12 days of age. Beginning at 5 - 7 weeks of age, mice were exposed to 0 or 2039 p.p.m. UG
vapor 6 h/day, 5 days/week for 13 weeks.
b
Assumes that 1 g liver •= 1 cm3.
°Fold increase over DEN/control group is inHirntH in brackets.
d
Significantly different from DEN/control group.
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Hepatic preneoplastic findings
At necropsy, pale white masses (generally <, 1 mm) were visible
in the livers in 2/8 mice in the DEN/control group and 7/8 mice
in the DEN/UG group (Table II). Microscopic examination of
H&E-stained liver sections was used to quantitate altered hepatic
foci which were found exclusively in mice initiated with DEN
(Table II). Over 96% of the foci were comprised of cells that
were darker blue, smaller and had greater vacuouzation than
hepatocytes in the surrounding liver and were therefore classified
as basophilic foci (16). It was noted that 8/94 basophilic foci
(8.5 %) appeared to protrude into the lumen of hepatic veins. In
addition, two foci were classified as eosinophilic and one as clear
cell (16). All foci phenotypes from a given animal were grouped
together for the purposes of stereological analysis.
gasoline vapor
A.M.Standeven and T.L.Goldsworthy
Table m . Comparative effects of PS-6 and API 9 1 - 1 blend UG on final body weight, relative liver weight , serum SDH activity, hepatocyte labeling index
and hepatic PROD activity in female B6C3F1 mice
Treatment*
n
Final
body wt
(g)
Liver
wt
(% body wt)
Serum
SDH
activity1'
Hepatocyte
labeling
index (%)
Hepatic
PROD
activity0
Experiment 1
Corn oil
PS-6
8
8
23.6 ± 0.7
24.1 ± 1.0
5.29 ± 0.28
6.92 ± G.&
16.4 ± 1.6
15.8 ± 2.6
2.7 ± 2.1
24.2 ± 8.2"1
35.4 ± 4.3
311.1 ± 51.9"1
Experiment 2
Corn oil
API 9 1 - 1
8
8
24.0 ± 0.7
24.5 ± 1.1
5.22 ± 0.39
6.41 ± 0.39"*
16.9 ± 3.2
15.1 ± 1.9
3.2 ± 4.4
22.8 ± 7.8"
21.2 ± 4.5
211.0 ± 41.9"1
treatment with PS-6 and API 9 1 - 1 blend UG produced similar
increases in hepatocyte labeling index (9.0- versus 7.1-fold
respectively), relative liver weight (31% versus 23%) and hepatic
PROD activity (8.8- versus 10.0-fold).
Discussion
An initiation—promotion protocol consisting of a single treatment
of infant B6C3F1 mice with DEN (24,25) and subchronic
treatment with UG vapor was used to test the hypothesis that
UG vapor acts as a liver tumor promoter in female mice. The
conditions of the UG exposure closely approximated those of the
cancer bioassay in which inhalation of UG vapor was shown to
be hepatocarcinogenic. We found that exposure of DEN-initiated
female B6C3F1 to 2039 p.p.m. UG vapor 6 h/day, 5 days/week
for 13 weeks resulted in significant increases (3- to 4- fold) in
the size and volume fraction of altered hepatic foci in these mice
relative to DEN-initiated controls. Such effects are hallmarks of
promotion (26) and establish that UG acts as a promoter of hepatic
preneoplastic lesions. Moreover, UG treatment failed to induce
altered hepatic foci in the absence of prior DEN treatment. This
observation is consistent with, but does not prove, the hypothesis
that UG lacks significant initiating activity.
Thorough studies of the progression of liver tumors in this
mouse model have shown that altered hepatic foci, in particular
the basophilic foci commonly produced by DEN treatment, are
almost certainly precursors of adenomas and carcinomas
(27-29). The altered hepatic foci promoted by UG in the present
study were predominantly basophilic, and a small fraction of these
(8.5%) appeared to protude into hepatic veins. Such venous
extension has been reported previously in this model (25,29),
though the significance of diis observation is unclear.
It is noteworthy that the promotional effect of UG was rather
weak as compared to other chemicals tested in this two-stage
model of carcinogenesis (30,31). PS-6 blend UG contains >40
major hydrocarbon components (2), which is typical of all
gasolines (32). Thus, we can only conclude that the complex
mixture that PS-6 blend UG represents was a weak liver tumor
promoter and cannot exclude the possibility that individual
components of UG may be more potent liver tumor promoters,
or that some components of UG inhibit tumor promotion. Such
components may be more or less prevalent in other blends of
gasoline.
The mechanism of liver tumor promotion by UG was not
specifically addressed in the present study. However, in
agreement with a recent study (9), exposure of female mice to
2039 p.p.m. UG vapor for 13 weeks caused a sustained increase
2140
in liver weight in the absence of hepatic necrosis. Moreover, the
mice gained weight normally over the exposure period (9; the
present study). Thus, it seems clear that UG was not hepatotoxic
or overtly toxic to mice under conditions in which it promoted
altered hepatic foci. Tilbury et al. (9) also have observed that
UG at the hepatocarcinogenic exposure concentration induces a
transient burst ( < 2 weeks) of hepatocyte proliferation. Taken
together, these data show that UG can be grouped with a class
of non-genotoxic hepatocarcinogens termed mitogens that
includes phenobarbital and 1,4-dichlorobenzene (10). Insights
gained into the mechanism of hepatocarcinogenicity of these latter
chemicals may therefore be relevant to liver tumor promotion
by UG.
Lubet and co-workers have noted an association between the
ability of some chemicals, mainly barbiturates and hydantoins,
to act as rat liver tumor promoters and their ability to induce
a pleiotropic response that includes induction of CYP2B (12,13).
However, it is not clear whether or not this correlation between
liver tumor promotion and CYP2B induction holds in the mouse
(33). The dealkylation of pentoxyresorufin appears to be specific
to CYP2B isoforms in the rat (20,34), and recent data suggest
that Cyp2b-10 is the major catalyst of PROD activity in female
BALB/c mice (35). Thus, PROD induction appears to be a
reliable marker for CYP2B induction. We therefore ascertained
if UG would induce PROD activity in female mice under
exposure conditions that resulted in tumor promotion. Indeed,
exposure of female B6C3F1 mice to 2039 p.p.m. of PS-6 blend
UG vapor for 13 weeks resulted in an -8-fold induction of
PROD activity. There is evidence, at least in rats, that the potency
of agents as liver tumor promoters correlates with the strength
of hepatic PROD induction by these agents (12). Thus, the
relatively weak induction of PROD activity by UG as compared
to other promoters (12) is consistent with the impression that UG
was a weak promoter in this system. Nevertheless, our data
extends the strong, but not perfect (33,36), correlation between
hepatic CYP2B induction and liver tumor promotion in rodents.
UG composition is highly variable between manufacturers and
over time (1). Since PS-6 blend UG was formulated over 15 years
ago (3), we wished to extend the above findings by comparing
acute effects of PS-6 blend UG and a more recent blend of UG,
API 91 - 1 , on hepatic endpoints that might be relevant to tumor
promotion by UG. We found that each blend of UG administered
i.g. for 3 days at 1800 mg/kg/day produced quantitatively similar
increases in relative liver weight (23 - 3 1 %), hepatocyte labeling
index (7- to 9-fold) and PROD activity (9- to 10-fold).
Significantly, these acute effects of oral UG treatment were
Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on September 16, 2016
•Mice were gavaged for three consecutive days with 1800 mg/kg/day PS-6 or API 9 1 - 1 blend UG in corn oil or corn oil alone.
b
Sigma units/1.
"THDOI resorufin/min/mg microsomal protein (n = 4, pooled from 2 mice/sample).
Significantly different from respective corn oil control.
Promotion of preneoplastic lesions by
gasoline vapor
qualitatively the same as subchronic exposure to ~2056 p.p.m.
PS-6 vapor (9; the present study). Thus, it is reasonable to suggest
that API 9 1 - 1 blend UG, which represents an 'industrial
average' of UG used in the United States in 1988 (14,15), is likely
to have similar promotional effects as PS-6 blend UG in female
mouse liver.
In conclusion, UG vapor is a relatively weak promoter of
hepatic preneoplastic lesions and induces hepatic CYP2B in
female mice under exposure conditions that resulted in liver
tumors in a cancer bioassay. Since it may be possible to find
a 'no-effect' level for this promotional effect of UG, these findings
may aid in the daunting task of extrapolating the
hepatocarcinogenic effect of UG in mice to a safe exposure level
in humans.
2141
Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on September 16, 2016
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Acknowledgements
phenobarbital and other xenobiotics in the rat. Arch. Biochem. Biophys., 238,
We thank Paul Ross, Ardie James, Patricia O'Brien-Pomerleau and Drs Byron
43-48.
Butterworth, Russel Cattley, Gregory Kedderis, Owen Moss, James Popp and
21.Matsubara,T., Koike.M., Touchi.A., Tochino.Y. and Sugeno.K. (1976)
Doug Wolf for helpful suggestions. We also thank Carol Bobbitt, Kathy Bragg,
Quantitative determination of cytochrome P-450 in rat liver homogenates.
Elizabeth Humphrey, Richard Masney, Steven Butler and Tim Shepard for
Anal. Biochem., 75, 596-603.
excellent animal care, Marianne Marshall, Carl Parkinson, Del Ponder and Kay
22.EMridge,S.R., Tilbury.L.F., Goldsworthy.T.L. and Butterworth,B.E. (1990)
Roberts for inhalation exposures, Corrie Dunn, Otis Lyght, Mary Morris and
Measurement of chemically induced cell proliferation in rodent liver and
Delorise Williams for necropsy assistance, and Mike Judge, Don Stedman and
kidney: a comparison of 5-bromo-2'-deoxyuridine and [3H]thymidine
Vooda Teets for technical assistance. This work was supported in part by a grant
administered by injection or osmotic pump. Carcinogenesis, 11,2245—2251.
from the American Petroleum Institute and NRSA 1F32ES05599-01 from the
23. International Research and Development Corporation (IRDC) (1983) Motor
National Institute of Environmental Health Sciences (A.M.S.).
Fuel Chronic Inhalation Study, Vol. 1-6. Sponsored by the American
Petroleum Institute.
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Received on March 16,1993; revised on May 14,1993; accepted on June 9,1993