Carcinogenesis vol.18 no.5 pp.1075–1083, 1997
Loss of tumor-promoting activity of unleaded gasoline in
N-nitrosodiethylamine-initiated ovariectomized B6C3F1 mouse
liver
Glenda J.Moser, Douglas C.Wolf, Brian A.Wong and
Thomas L.Goldsworthy1
Chemical Industry Institute of Toxicology, Research Triangle Park, NC
27709, USA
1To
whom correspondence should be addressed
Unleaded gasoline (UG) vapor (2056 ppm) increased the
incidence of liver tumors in a chronic bioassay and exhibited
tumor-promoting activity in N-nitrosodiethylamine (DEN)initiated female mouse liver. Estrogen inhibited mouse liver
tumor development and the hepatocarcinogenic and tumorpromoting dose of UG produced uterine changes suggestive
of estrogen antagonism. To directly test the hypothesis that
UG-induced tumor-promoting ability is secondary to its
interaction with the mouse liver tumor inhibitor, estrogen,
we compared the tumor-promoting ability of UG in ovariectomized (Ovex) mice with the hepatic tumor-promoting
ability of UG in intact mice. Ovaries were surgically
removed at 4 weeks of age. Exposure to wholly vaporized
UG (2018 ppm) under bioassay and tumor-promoting
conditions began at 8 weeks of age. After 4 months of
exposure, UG increased relative liver weight and hepatic
microsomal cytochrome P450 pentoxyresourfin-O-dealkylase and ethoxyresorufin-O-deethylase activity to a similar extent in intact and Ovex mice. Non-focal hepatocyte
proliferation, as measured by the incorporation of bromodeoxyuridine, was not changed by UG exposure and was
similar in all treatment groups. After 4 months of exposure
to DEN-initiated mice, UG significantly increased the volume fraction of liver occupied by foci (three-fold) as
compared to control intact mice. As expected, volume of
foci was elevated in DEN/Ovex/control mice as compared
to DEN/intact/control mice. In DEN/Ovex mice UG did not
significantly increase the focal volume fraction. Thus, the
tumor promoting activity of UG, as demonstrated by
increased volume fraction of liver occupied by hepatic foci
in intact mice, is greatly attenuated in Ovex mice. The
volume fraction data in Ovex mice support the hypothesis
that the tumor promoting activity of UG is dependent upon
the interaction of UG with ovarian hormones. These data
also indicate that hepatic microsomal cytochrome P450
PROD and EROD induction, hepatomegaly and non-focal
hepatic LI are not specific markers of hepatic tumor
promoting activity of UG.
Introduction
The public is annually exposed to vapors from billions of
gallons of unleaded gasoline (UG*). The major route of
exposure is by inhalation to workers during the production
*Abbreviations: UG, unleaded gas; DEN, N-nitrosodiethylamine; BrdU, anti5-bromo-29-deoxyuridine; ALT, alanine aminotransferase; LI, labeling index;
PROD, 7-pentoxyresorufin-O-dealkylase; EROD, 7-ethoxyresorufin-O-deethylase.
© Oxford University Press
and transportation of UG and to the general public during
refueling at service stations (1). The human health risk from
intermittent, low-dose exposure to UG vapor is unknown.
In a 2-year cancer bioassay in both sexes of mice and rats,
the high dose of 2056 ppm, but not lower doses of 67 ppm
and 292 ppm of a reference formulation of UG (PS-6)
selectively increased the incidence of liver tumors in female
mice (2). The significance of the species- and sex-specific
response is unknown. However, sex hormones have long been
known to play a role in mouse hepatocarcinogenesis. In studies
of carcinogenesis, male mice are generally found to have a
higher incidence of spontaneous and chemically-induced liver
tumors than female mice (3,4). Gonadectomy of male mice
decreased and ovariectomy of female mice increased the
incidence of tumors in spontaneous and N-nitrosodiethylamine
(DEN)-initiated liver (5). Furthermore, estrogen inhibited the
growth of hepatic foci in DEN-initiated male and female mice
(6,7). Thus, a role for sex hormones in hepatocarcinogenesis
is well established.
In a two-stage, initiation–promotion model system in DENinitiated female mouse liver, exposure to PS-6 UG vapor for
4 months increased the size of hepatic foci and volume fraction
of the liver occupied by foci relative to DEN/controls. The
increased growth of hepatic foci occurred only at the hepatocarcinogenic dose of 2038 ppm (7,8). In addition, PS-6 UG
was negative in short-term in vitro tests for genetic toxicity
(9–13). Together, these data suggest that PS-6 UG is hepatocarcinogenic through a tumor-promoting mode of action. Formulations of UG change with time and manufacturer. We previously
determined that a blend of UG representative of that used in
1991 UG 91-01 produced short-term effects after inhalation
and gavage similar to those produced by the hepatic tumor
promoter PS-6 UG (7,16). To determine if the newer formulation of UG had tumor promoting ability in intact mice, we
conducted an initiation-promotion experiment utilizing 9101 UG.
After short-term, subchronic and chronic exposure, UG
produced biological responses suggestive of endocrine modulation (7,14–16). In the chronic bioassay, the hepatocarcinogenic
dose of PS-6 UG, but not lower non-hepatocarcinogenic doses,
decreased uterine endometrial cystic hyperplasia and increased
uterine atrophy (14). After short-term exposure by gavage or
inhalation, and after subchronic inhalation exposure to the
high dose of 2038 ppm, UG decreased uterine weight (7,15,16).
Since endometrial hyperplasia and uterine weight are for the
most part under the control of estrogen, UG exhibited effects
consistent with estrogen antagonism.
Given that estrogen inhibits mouse liver carcinogenesis
and that UG exhibits antiestrogenic-like effects, we have
hypothesized that the hepatic tumor-promoting ability of UG
is secondary to its interaction with the ovarian hormone
estrogen. To directly test this hypothesis, we examined the
effect of ovarian hormones on the hepatic tumor-promoting
ability of UG and on indicators related to hepatic tumor
1075
G.J.Moser et al.
promotion. This was done by comparing the effects of UG in
ovariectomized (Ovex) mice to the responses of UG in intact
mice. Here, we demonstrate that the newer formulation of UG
is also a hepatic tumor promoter in intact mice, and the tumor
promoting activity of UG is greatly attenuated in the absence
of ovarian hormones.
Materials and methods
Chemicals
91-01 UG was provided by the American Petroleum Institute (Washington,
DC). As compared with PS-6 UG, the 91-01 blend of UG contains a slightly
greater percentage of aromatics (33.2% versus 26.1%) and olefins (12.5%
versus 8.4%), and a lower percentage of saturated hydrocarbons (53.1%
versus 65.5%) (8). Monoclonal anti-5-bromo-29-deoxyuridine (BrdU) for
immunohistochemistry was purchased from Becton-Dickson (Mountain View,
CA). Isoflurane was obtained from Anaquest (Guayama, Puerto Rico). All
other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
Animals
All experiments were conducted under National Institutes of Health (NIH)
guidelines for the care and use of laboratory animals and approved by the
Institutional Animal Care and Use Committee, Chemical Industry Institute of
Toxicology (CIIT). To breed B6C3F1 mice, male C3H/HeNCrlBR mice and
female C57BL/6NCrlBR mice were obtained from Charles River Breeding
Laboratories (Raleigh, NC). All mice were quarantined upon arrival for at
least 10 days and housed in humidity- and temperature-controlled HEPAfiltered rooms in facilities accredited by the American Association of Laboratory Animal Care.
Experimental design
A one-factor (UG treatment), two-level design was used to evaluate the
hepatic tumor-promoting ability and subchronic hepatic responses of UG.
Since the purpose of the experiment was to compare the hepatic tumorpromoting ability of UG in Ovex mice to the tumor-promoting ability of UG
in intact mice, the effects of ovariectomy and DEN-initiation were blocked
in this experiment. The following outcomes were evaluated: number and size
of hepatic foci, volume fraction of the liver occupied by foci, number
of macroscopic hepatic lesions, liver weight, microsomal P450 activity,
hepatotoxicity as measured by the liver enzyme alanine aminotransferase
(ALT), liver DNA synthesis or labeling index (LI) as measured by the
incorporation of 5-bromo-29-deoxyuridine, and length and stage of the estrous cycle.
Animal treatments
B6C3F1 mice were bred as previously described (8). Twelve-day-old female
B6C3F1 mice were initiated between 12:00 p.m. and 2:00 p.m. with a single
i.p. injection of DEN (5 mg DEN/kg, 7.1 ml/kg body wt) or saline in
accordance with the infant mouse initiation–promotion model system (5,7,8).
After weaning at ~28 days of age, mice randomly assigned by body weight
were surgically Ovex or ovaries were left intact. For bilateral Ovex, ovaries
were visualized by a dorsal midline incision caudal to the last rib, clamped
at the level of the oviducts and excised.
Beginning at 8 weeks of age, groups of 12 DEN-initiated and salineinjected mice that had been ovariectomized or that had intact ovaries were
exposed to control air or a target concentration of 2038 ppm 91-01 UG.
Exposure conditions and dosing regimens were as previously described
(Figure 1) (8,14). Intact and Ovex mice were each divided into four groups:
saline/control (non-initiated), saline/UG (UG control), DEN/control (initiation
control) and DEN/UG (initiation–promotion). Intact mice exposed to UG were
housed in 1-m3 chambers in a 8-m3 chamber. Ovex mice exposed to UG were
housed in 1-m3 chambers in a separate 8-m3 chamber. Chamber concentrations
were determined at least hourly by injecting samples from the chamber into
a Hewlett-Packard 5720A gas chromatograph. Standard calibration curves
were generated by injecting a known volume of UG into a known volume of
air in a Tedlar bag. Calculations in parts per million were made by utilizing
an average molecular weight of 94.3 g/mol and density of 0.743 g/ml. Control
groups were housed in identical inhalation chambers and exposed to Hepafiltered particulate-free outside air that contained 0 ppm UG. One group
(DEN/UG/reversal) of DEN/intact/UG mice was exposed to UG vapor for 4
months and then exposed to control air for 4 months. Exposures were
carried out during the light hours (8:30 a.m.–2:30 p.m.) on week days,
including holidays.
Filter-purified tap water and NIH-07 pelleted diet (Ziegler Bros., Gardners,
PA) were available ad libitum, except that feed was not available during the
6-h inhalation exposure periods. Water and food were analysed for pesticide
1076
Fig. 1. Treatment protocol. Twelve-day-old female B6C3F1 mice were
initiated with a single i.p. injection of DEN (5 mg DEN/kg, 7.1 ml/kg body
wt) or saline and exposed to control air or UG for 4, 7 or 8 months. The
DEN/UG reversal group was exposed to 2000 6 80 ppm UG for 4 months
followed by control air for 4 months.
contamination, and food was evaluated for nutritive content. The chambers
were maintained at 68–76°F and 40–60% humidity and on a 12-h light-dark
cycle with the light period extending from 7:00 a.m. to 7:00 p.m. Mice were
randomized by computer-generated numerical randomization tables by weight
before the start of exposure so that mean starting weights of the groups were
not significantly different. Mice were observed at least twice daily on UGexposure days and at least once daily on days when not exposed to UG.
Clinical observations were recorded at least once weekly. Body weights were
determined at least once every 2 weeks. The virus-free status of mice was
confirmed monthly by standard serological analysis on sentinel mice that were
housed with their respective treatment groups.
Necropsy
Mice were weighed and anesthetized with isoflurane ~18 h after the last
exposure. The thoracic cavity was opened and blood withdrawn via cardiac
puncture with a 21-gauge needle. Mice were euthanized by exsanguination.
Blood was allowed to clot for a minimum of 30 min before centrifugation to
obtain serum for ALT. Vaginal smears were taken at the time of necropsy by
making impression smears of excised vagina. Ovex status was confirmed at
the time of necropsy. Uterus, ovaries if present, adrenals and pituitary were
removed, trimmed of extraneous tissue, rinsed in saline, blotted dry, examined
and weighed. Uterine fluid was not expressed from uteri before weighing.
Livers were removed, rinsed in saline and blotted dry. After weighing, the
liver was separated by lobes, sectioned into 2-mm strips and examined for
the presence of macroscopically visible lesions. All observed macroscopic
lesions in the liver were described, mapped, and quantitated by size (ø 2, 2
, X ø 4, or . 4 mm). No hepatic lesions .4 mm were found at 4 months.
Liver sections from the left, median right and anterior lobes along with a
section of duodenum, uterus containing the uterine body, and one uterine
horn, adrenals, pituitaries and ovaries were fixed in 10% phosphate-buffered
formalin for 48 h and then transferred to 70% ethanol. Tissues were embedded
in paraffin by routine methods and sectioned at 5 µm, stained with hematoxylin
and esoin (H&E), and examined by light microscopy. Remaining liver was
minced, rinsed, and either used to prepare microsomes or flash-frozen.
Remaining uterus was flash-frozen. Frozen liver and uterus were stored
at –80°C.
Microsome preparation
At necropsy, livers from at least three mice within the same group were
flushed and microsomal preparations were made by standard methods of
differential centrifugation as previously described (17). Microsomes were
stored at –80°C.
Microsomal assays
Hepatic microsomal cytochrome P450 7-pentoxyresorufin-O-dealkylase
(PROD) and 7-ethoxyresorufin-O-de-ethylase (EROD) activity from at least
three different samples within each group were determined as previously
described (8). PROD is a marker enzyme of the cytochrome P450 2B1/2
family and EROD is a marker enzyme of the P450 1A1/2 family. The
fluorometric method of Lubet was used to determine PROD and EROD
activity (18). The rate of increase in fluorescence was converted to pmol
resorufin per minute by means of a standard curve generated from the
fluorescence of known amounts of resorufin. The assays were linear with time.
Tumor-promoting ability of unleaded gasoline
Microsomal protein was assayed with Coomassie-Plus Protein Assay
Reagent (Pierce, Rockford, IL) using bovine serum albumin (Pierce) as
a standard.
ALT
Serum ALT activity was assayed on the same day as the necropsy using a
commercial kit (no. 44924) from Roche Diagnostics and a Roche Cobas Farra
II clinical analyser.
Hepatocyte proliferation
At ~3:00 p.m. three-and-a-half days before necropsy, mice were implanted
subcutaneously with 7-day osmotic pumps (Alzet model 2001, 1.06 µl/h, Alza
Corporation, Palo Alto, CA) containing 16 mg BrdU (Sigma Chemical Co.,
CAS no. 59-14-3, .99% pure)/ml phosphate-buffered saline (pH 7.2) without
magnesium. Coded paraffin sections of liver were immunohistochemically
stained for BrdU (19) and non-focal hepatocyte DNA synthesis was determined
as previously described by light microscopy with the investigator blind as to
animal treatment (8). BrdU incorporation in duodenum on the same tissue
sections was employed as a positive control for immunohistochemical staining
and for delivery of BrdU to the mouse. The minimal total number of nonfocal hepatocellular nuclei counted was 1000 nuclei from the left lobe of
livers. Hepatic LI were calculated by dividing the number of labeled nuclei
by the total number of hepatocellular nuclei counted and multiplying by
100. The mean 6 SD from each group was calculated after tissue slides
were decoded.
Vaginal smears and vaginal lavages
Vaginal smears were taken at necropsy by impressing the vagina onto a glass
slide. For vaginal lavages, the vagina was flushed with two drops of
physiological saline and aspirated back into the tip of a glass eye dropper.
The flush aspirate containing cells washed from the vaginal wall was placed
onto a glass slide. Both necropsy smears and vaginal lavage aspirates
were air-dried, stained with Wrights stain, and examined microscopically to
determine the stage of estrous at the time the sample was taken. The
predominance or relative ratio of nucleated epithelia, cornified, squamous
epithelia and leukocytes was used to indicate the stage of estrous as early
proestrus, late proestrus, estrus or diestrus. Vaginal lavages were done at
~7:00 a.m. prior to the start of exposure for at least 10 consecutive days.
Quantitation of altered hepatic foci
The total area of all three liver sections was determined with an Image-1
image processing system (Universal Imaging Corp., West Chester, PA). With
the experimenter blind to the treatment group, hepatic foci ù10 cells in
size were located and phenotypically classified as basophilic or acidophilic
according to published criteria (20). The area of individual hepatic foci was
used to calculate the number of foci per cm3, number of foci per liver, mean
volume of foci, and volume fraction of the liver occupied by foci according
to the sterological analysis of Pugh et al. (21).
Statistical analysis
Each group consisted of 10–12 animals. Mean 6 SD was determined for each
of the following quantitative end points: body, liver, uterus, ovary, adrenal
and pituitary weight (g), relative organ weight (organ-to-body wt ratio
expressed as %); serum ALT (IU/l); PROD activity (pmol/min/mg microsomal
protein); EROD activity (pmol/min/mg microsomal protein); hepatic LI (%);
number of macroscopic lesions by size categories; number of hepatic foci
(per cm3 or liver); volume fraction of liver occupied by hepatic foci (%); and
mean hepatic volume (mm3). The UG treatment group means were compared
to intact and Ovex control means by the unpaired, two-sided Student’s t-test.
Data from the hepatic LI, mean volume of foci and volume fraction of the
liver were transformed since they were not normally distributed. LI data was
transformed by arcsin of the square root of LI, and foci size and volume
fraction were log-transformed before testing for significance by Student’s ttest. Fisher’s Exact Test was utilized to determine if mice exposed to UG
produced an estrous cycle profile different from that of controls. After
determining that standard deviations were not equal in control and UGexposed mice, Welch Anova was utilized to evaluate vaginal lavage data. A
significance level of 0.05 was used for all analyses.
Results
Exposure conditions and survival
The mean UG exposure concentrations and standard deviations
at 4 months were 2000 6 80 ppm for intact mice and 2021 6 72
ppm for Ovex mice (data not shown). These duration-adjusted
exposure levels closely approximated the target exposure
concentration of 2038 ppm UG (98.9% and 99.2%, respect-
ively). The only treatment related observation in B6C3F1 mice
during subchronic UG exposure was occasional hypoactivity.
At 4 months, survival was 100% for saline/intact/controls,
92% (11/12) for DEN/intact/controls, 83% (10/12) for saline/
intact/UG, 92% (11/12) for DEN/intact/UG, 100% for saline/
Ovex/controls, 100% for DEN/Ovex/controls, 92% (11/12) for
Saline/Ovex/UG, and 100% for DEN/Ovex/UG.
Body and organ weights
UG, Ovex, or DEN did not significantly alter body weight in
any treatment groups (Table I). UG significantly increased
absolute (data not shown) and relative liver weight between
25% and 33% (Table I). The relative uterine weight at 4
months decreased in all intact mice after UG exposure. UG
exposure decreased ovary weight in both saline-treated and
DEN-initiated mice, but the decrease was statistically significant only in DEN-initiated mice. In intact mice, UG significantly
decreased relative pituitary weight 28 and 17% in salinetreated and DEN-initiated mice, respectively (Table I). UG did
not significantly alter adrenal weight in intact or Ovex mice
(data not shown).
Histological evaluations
Histopathological examination of H&E-stained livers by light
microscopy indicated that DEN treatment did not produce
detectable nonneoplastic histological changes. Livers from
mice exposed to UG in saline/intact, DEN/intact, saline/Ovex,
and DEN/Ovex groups showed mild centrilobular to midzonal
hypertrophy involving approximately one-half to two-thirds of
the lobule without hepatic necrosis. The liver specific enzyme,
ALT, was not elevated in any treatment group (data not shown).
The female reproductive tracts were not different microscopically after UG treatment. Uterine endometrial cysts were
present in all controls and 80% of UG-exposed mice. Mice in
all treatment groups had corpa lutea and developing follicles.
Vaginal smears taken at the time of necropsy suggested no
statistically significant difference between intact saline-treated
and DEN-initiated mice. Thus the data from saline/intact/
controls and DEN/controls, and saline/UG and DEN/UG mice
were combined to increase statistical power. Evaluation of
vaginal smears taken at the time of necropsy demonstrated a
significant difference in the percentage of mice in the various
stages of estrous after UG exposure as compared to non-UGexposed mice (Table II).
Non-neoplastic hepatic effects
Hepatic microsomal hepatic P450 PROD and EROD activities
were significantly increased in livers from saline/UG, DEN/
UG, saline/Ovex/UG and DEN/Ovex/UG mice as compared
to their respective controls (Table III). At 4 months, there were
no significant differences in non-focal hepatic LI in any
treatment groups as compared to controls (Table III).
Hepatic foci evaluations
Identification, location, and the phenotype of individual hepatic
foci were determined by microscopic examination of H&Estained liver sections. At 4 months, no hepatic foci were found
in saline-treated mice (data not shown). UG did not significantly
alter the number of hepatic foci per cm3 or per liver in DENinitiated intact or Ovex mice (Table IV). In DEN-initiated
intact mice, UG significantly increased the mean size of foci
6.6-fold as compared to the DEN/intact/control group. As
expected, there was a significant increase in the size of hepatic
foci in DEN/Ovex mice as compared to DEN-initiated intact
mice. UG only slightly increased the size of foci in DEN/
1077
G.J.Moser et al.
Table I. Body weight and relative organ weight of intact and ovariectomized B6C3F1 female mice exposed to UG for 4 months
a, b
Group
Body weight (g)
Liver (%)
Uterus (%)
Ovary (3102 %)
Pituitary (3103 %)
Saline/control
Saline/UG
DEN/control
DEN/UG
Saline/Ovex/control
Saline/Ovex/UG
DEN/Ovex/control
DEN/Ovex/UG
36.8 6 4.3
38.7 6 3.7
36.0 6 4.9
38.5 6 4.0
38.5 6 2.5
41.1 6 3.1
38.9 6 4.2
39.7 6 3.7
4.5 6 0.3
5.6 6 0.5*
4.4 6 0.5
5.8 6 0.3†
4.2 6 0.3
5.5 6 0.7‡
4.5 6 0.6
5.9 6 0.5§
0.524 6 0.210
0.277 6 0.086*
0.520 6 0.165
0.344 6 0.086†
0.067 6 0.041
0.059 6 0.019
0.065 6 0.028
0.044 6 0.018
3.05 6 0.73
2.75 6 0.51
3.40 6 0.97
2.41 6 0.55†
8.4 6 2.4
6.0 6 0.6*
7.8 6 2.3
6.5 6 1.5†
5.2 6 2.2
5.0 6 1.1
5.7 6 1.3
5.8 6 1.3
aTwelve-day-old
female B6C3F1 mice were initiated with a single i.p. injection of DEN. Beginning at 8 weeks of age, groups of 10–12 mice were exposed to
2000 6 80 UG ppm for intact mice and 2021 6 72 UG ppm in Ovex mice for 4 months.
bMean 6 SD of 10–12 mice.
*Significantly different from saline/intact/control (P , 0.05).
†Significantly different from DEN/intact/control (P , 0.05).
‡Significantly different from saline/Ovex/control (P , 0.05).
§Significantly different from DEN//Ovex/control (P , 0.05).
Table II. Effect of UG exposure for 4 months on stage of estrous cycle at
time of necropsy in B6C3F1 micea
Group
Diestrus
(%)
Early proestrus
(%)
Late proestrus
(%)
Estrus
(%)
Controls
UG-exposed*
0
12
17
41
22
18
61
29
aTwelve-day-old
female B6C3F1 mice were initiated with a single i.p.
injection of DEN. Beginning at 8 weeks of age, groups of 10–12 mice were
exposed to UG for 4 months. At the time of necropsy, vaginal smears were
taken, air-dried, stained with Wrights stain, and examined microscopically.
*Significantly different from controls (P , 0.05).
Table III. Hepatic microsomal cytochrome P450 PROD and EROD activity
and hepatocyte labeling index in intact and ovariectomized B6C3F1 female
mice exposed to UG for 4 monthsa
Group
Saline/control
Saline/UG
DEN/control
DEN/UG
Saline/Ovex/control
Saline/Ovex/UG
DEN/Ovex/control
DEN/Ovex/UG
PROD
(pmol/min/mg)b
EROD
Hepatic labeling
(pmol/min/mg)b index (%)c
23 6 6
51 6 24
25 6 5
117 6 15†
17 6 7
166 6 7‡
22 6 6
87 6 21§
47 6 21
182 6 38†
69 6 25
227 6 54‡
39 6 28
97 6 16§
0.20 6 0.22
0.1560.19
0.13 6 0.09
0.23 6 0.19
0.19 6 0.29
0.07 6 0.10
0.06 6 0.06
0.19 6 0.16
aTwelve-day-old
female B6C3F1 mice were initiated with a single i.p.
injection of DEN. Ovex mice were surgically ovariectomized at 28 days of
age. Beginning at 8 weeks of age, mice were exposed to UG for 4 months.
Microsomes were prepared at the time of necropsy.
bMean 6 SD of at least three microsomal samples.
cHepatic LI was determined with three-and-a-half-day osmotic pumps
containing 16 mg BrdU/ml.
†Significantly different from DEN/intact/control (P, 0.05).
‡Significantly different from saline/Ovex/control (P , 0.05).
§Significantly different from DEN/Ovex/control (P , 0.05).
Ovex/UG mice as compared to DEN/Ovex/control mice (1.8fold). In DEN-initiated intact mice, UG exposure significantly
increased the volume fraction of the liver occupied by foci
3.5-fold as compared to DEN/intact/control mice. In DEN/
Ovex/UG mice, UG did not significantly increase the focal
volume fraction as compared to DEN/Ovex/control mice (1.2fold) (Table IV).
1078
Hepatic neoplastic effects
At 4 months only one mouse from the DEN/intact/control
group had macroscopic hepatic lesions. The incidence of
macroscopic lesions in DEN/intact/UG mice was 82% with a
significant increase in the number of macroscopic lesions per
mouse (Figure 2). We also categorized the macroscopic lesions
according to size. UG exposure in DEN-initiated intact mice
predominantly increased the number of lesions ø2 mm in size
and also significantly increased the number of lesions between
2 and 4 mm in size (Figure 2). Macroscopic lesions were
found to be hepatocellular adenomas and carcinomas.
In DEN/Ovex mice all except one DEN/Ovex/control mouse
had hepatic macroscopic lesions. The number of tumors per
mouse was not significantly different in DEN/Ovex/UG mice
as compared to DEN/Ovex/control mice. Additionally, there
was no significant difference in the percentage of lesions in
each of the size categories in the DEN/Ovex/control and DEN/
Ovex/UG mice (Figure 2).
Seven- or eight-month exposures
An additional 10–12 mice in saline/control, saline/UG, DEN/
control and DEN/UG groups were exposed for four more
months (total of 8 months) to further characterize the changes
seen in vaginal smears at 4 months (Figure 1). Twelve DENinitiated mice were also put into a DEN/UG/reversal group;
they were exposed to UG for 4 months followed by control
air for 4 months. Furthermore, groups of saline/Ovex/control,
saline/Ovex/UG, DEN/Ovex/control and DEN/Ovex/UG were
exposed for three more months or a total of 7 months to
evaluate continued effects of UG on uterine weight. The time
point of 7 months was chosen because of subtle losses of body
weight in DEN/Ovex mice.
The duration-adjusted exposure concentrations of UG were
2018 6 76 ppm for Ovex mice at 7 months and 2000 6 87
ppm for intact mice at 8 months (data not shown). Survival
was 92% (11/1/2) for saline/controls, 100% for saline/UG,
100% for DEN/controls, 92% (11/1/2) for DEN/UG, 100% for
DEN/UG/reversal, 100% for saline/Ovex/controls, 92% (11/1/
2) for saline/Ovex/UG, 100% for DEN/Ovex/controls and 92%
(11/12) for DEN/Ovex/UG mice.
The effects of UG exposure for 7 or 8 months as compared to
controls on body and relative organ weights and histopathology
were generally similar to those seen at 4 months. There were
no significant differences in body weight after UG exposure
Tumor-promoting ability of unleaded gasoline
Table IV. Parameters of hepatic foci in DEN-initiated intact and ovariectomized B6C3F1 female mice exposed to UG for 4 monthsa
Animal
DEN/Ct
DEN/UG
DEN/Ovex/Ct
DEN/UG/Ovex
Density
No./cm3
No./Liverb
Mean volume
(mm3)
Volume fraction
(%)
301 6 47
253 6 34
391 6 48
267 6 28
500 6 68
507 6 68
690 6 112
583 6 53
3.15 6 0.71
20.94 6 6.92*
15.21 6 2.68
28.00 6 7.56†
1.20 6 0.21
4.17 6 1.03*
5.43 6 1.08
6.59 6 1.56
aTwelve-day-old
female B6C3F1 mice were initiated with a single i.p. injection of DEN. Ovex mice were surgically ovariectomized at 28 days of age.
Beginning at 8 weeks of age, mice were exposed to UG mice for 4 months.
bAssumes 1 g liver 5 1 cm3.
*Significantly different from DEN/intact/control.
†Significantly different from DEN/Ovex/control.
Table V. Body weight and relative organ weight after UG exposure for 7 or 8 monthsa, b
Group
Body
weight (g)
Liver
(%)
Uterus
(%)
Ovary
(3102%)
Pituitary
(3103%)
Saline/control
Saline/UG
DEN/control
DEN/UG
DEN/UG/reversal
Saline/Ovex/control
Saline/Ovex/UG
DEN/Ovex/control
DEN/Ovex/UG
39.9 6 7.0
39.2 6 3.4
40.6 6 3.8
38.3 6 1.7
41.6 6 2.8
41.7 6 4.0
41.6 6 3.8
39.9 6 2.8
40.7 6 2.9
4.2 6 0.4
5.6 6 0.4*
6.3 6 1.4
11.6 6 3.7†
9.9 6 3.4
4.2 6 0.3
5.7 6 0.3§
9.2 6 3.1
12.2 6 3.6¶
0.738 6 0.325
0.294 6 0.063*
0.615 6 0.300
0.223 6 0.041†
0.410 6 0.117‡
0.236 6 0.091
0.105 6 0.054§
0.080 6 0.044
0.031 6 0.012¶
2.30 6 0.63
1.51 6 0.40*
2.35 6 0.60
1.67 6 0.31†
2.07 6 0.34‡
6.5 6 2.4
5.7 6 1.5
6.8 6 1.9
5.8 6 1.5
6.0 6 1.4
4.9 6 1.5
5.8 6 1.3
4.8 6 1.2
4.0 6 1.2
aTwelve-day-old
female B6C3F1 mice were initiated with a single i.p. injection of DEN. Beginning at 8 weeks of age, intact mice were exposed to
2000 6 87 ppm for 8 months and Ovex mice were exposed to 2018 6 76 ppm for 7 months.
bMean 6 SD of 10–12 mice.
*Significantly different from saline/intact/control (P , 0.05).
†Significantly different from DEN/intact/control (P , 0.05).
‡Significantly different from DEN/UG (P , 0.05).
§Significantly different from saline/Ovex/control (P , 0.05).
¶Significantly different from DEN/Ovex/control (P , 0.05).
Fig. 2. Effect of UG exposure for 4 months on incidence and mean number
and size of hepatic macroscopic lesions. Twelve-day-old female B6C3F1
mice were initiated with a single i.p. injection of DEN (5 mg DEN/kg, 7.1
ml/kg body wt) or saline. Beginning at 8 weeks of age, intact mice were
exposed to 2000 6 80 ppm UG for 4 months. Ovex mice were exposed to
2021 6 72 ppm UG for 4 months. Exposures were 6 h/day, 5 days/week.
Mice were killed ~18 h after the last exposure. At necropsy, the liver was
cut into 2-mm sections and examined for macroscopic lesions. The number
of lesions in each of three size classes (ø2 or 2 , X ø 4) was determined
for each animal, and the mean 6 SD was calculated for each group.
in any groups as compared to their appropriate controls
(Table V). Relative liver weight significantly increased after
UG-exposure for 7 or 8 months, with the greatest increase
occurring in DEN/UG mice as compared to the DEN/control
group (84%). UG-exposed livers demonstrated liver centrilobular to mid-zonal hypertrophy. Uterine weight significantly
decreased between 59 and 65% after continuous UG exposure
in all groups. Relative ovary weight significantly decreased 34
and 29% in saline/intact/UG and DEN/intact/UG mice as
compared to their respective intact controls. No histopathological differences in the female reproductive tract of UG-exposed
mice were found. There was no significant changes in adrenal
or pituitary weight after 7 or 8 months of UG exposure. In
DEN/UG/reversal mice, there was a decrease in relative liver
weight, and significant increase in relative uterus and ovary
weight relative to DEN/UG intact mice that had been continuously exposed to UG for 8 months.
Vaginal smears taken at the time of necropsy showed that
UG exposure produced significant changes in the percentage
of mice in the various stages of the estrous cycle as compared
to control mice irrespective of DEN-initiation (Table VI).
These differences in the estrous cycle were similar to those
seen after 4 months of UG exposure. Additionally, vaginal
lavages were done for a minimum of 10 days immediately
prior to necropsy. Vaginal lavage data supported the vaginal
smear data and indicated that UG exposure significantly
increased the length of the estrous cycle in both saline-treated
and DEN-initiated mice. Changes in the length of the estrous
cycle were due to significant increases in the number of days
in both the estrus and non-estrus stages (Table VI). Modulations
in the length of the estrous cycle were reversible. After 4
months of UG exposure followed by 4 months of exposure to
control air, the length of the estrous cycle and the mean time
1079
G.J.Moser et al.
Table VI. Effect of continuous UG exposure for 8 months on stages and
length of estrous cyclea in intact B6C3F1 mice
A
Group
Diestrus
(%)
Early proestrus Late proestrus
(%)
(%)
Estrus
(%)
Controls
UG-exposed*
12
38
6
24
49
21
33
17
Cycle length
(days)
Estrus length
(days)
Non-estrus length
(days)
4.6 6 0.7
6.0 6 0.8*
5.0 6 0.7
1.3 6 0.3
2.1 6 0.4*
1.5 6 0.5
3.2 6 0.3
3.9 6 0.4*
3.5 6 0.5
B
Controls
UG-exposedb
UG-reversalc
aTwelve-day-old
female B6C3F1 mice were initiated with a single i.p.
injection of DEN. Beginning at 8 weeks of age, groups of 12 intact mice
were exposed to UG for 8 months. At the time of necropsy, vaginal smears
were taken, air-dried, stained with Wrights stain and examined
microscopically.
bUG-exposed group includes saline/UG and DEN/UG mice.
cUG-reversal groups includes mice that were exposed to UG for 4 months
and then control air for 4 months.
*Significantly different from controls (P , 0.05).
in the estrus and non-estrus stages were similar to that of
DEN/control mice.
Discussion
The objective of this study was to determine if UG has tumor
promoting activity in the absence of ovarian hormones and to
establish potential mechanisms for UG-induced liver tumorigenesis. The two-stage initiation–promotion model of hepatocarcinogenesis results in the development of hepatic foci which
are putative preneoplastic neoplasms (22). Focal development
and growth are believed to be predictive of hepatocarcinogenesis (23). In the presence of the ovaries, UG had tumor
promoting activity. In Ovex mice, UG produced minimal
increases in focal size and volume fraction of the liver occupied
by hepatic foci. In accordance with the focal data, an increased
incidence and mean number of macroscopic lesions per mouse
were found in DEN-initiated intact mice exposed to UG at 4
months. In contrast, in DEN-initiated Ovex mice, UG exposure
did not significantly increase the incidence of mice harboring
macroscopic lesions or the number or size of macroscopic
lesions per mouse. Thus, in the absence of the ovaries, UG
has no significant tumor promoting activity suggesting a role
for ovarian hormones in the tumor promoting activity of UG
Sex hormones can modulate mouse liver tumorigenesis (5).
Therefore, it was not surprising that DEN/Ovex/control mice
demonstrated an increase in mean hepatic focal size (5-fold)
and an increased incidence and number of macroscopic lesions
(2-fold) as compared to intact DEN/control mice. The increase
in liver lesions in Ovex mice confirms previous observations
that ovarian hormones, probably estrogen, inhibit mouse liver
carcinogenesis (5–7). Mice were initiated 2 weeks before they
were ovariectomized. Since the majority of the DEN-initiated
DNA adducts are generally short-lived (24), the effect of Ovex
was most likely on the promotion of tumors. Because of high
Ovex control values it is possible that in the absence of ovarian
hormones, focal size, volume fraction of the liver occupied by
1080
foci and tumor development were already maximal so that UG
could have no effect on tumor promoting activity in Ovex
mice. However, others have demonstrated a mean foci size
over five-fold greater than that exhibited by DEN/Ovex/control
mice here (6,25–29). Furthermore, we and others have also
identified groups of Ovex mice with an average of over 40
macroscopic lesions per mouse (27,28; personal observation).
The significant decrease in uterine weight at 8 months in
saline/Ovex/UG and DEN/Ovex/UG mice relative to their
controls further suggests that Ovex mice had not reached their
maximal response in the uterus at 4 months. Thus, our hepatic
foci and macroscopic lesion data support the belief that foci
are the precursors to macroscopic hepatic lesions. In addition,
these studies suggest that at 4 months the inability of UG in
Ovex mice to increase volume fraction of liver occupied by
foci and number of macroscopic lesions is not because the
focal and tumor responses were maximal, but rather the tumor
promoting activity of UG is greatly attenuated in the absence
of ovarian hormones.
The ovaries secrete a number of hormones, including
estrogen (30). Because estrogen inhibits mouse liver tumorigenesis in female mice (6,7), we hypothesized that the hepatic
tumor-promoting ability of UG was secondary to its interaction
with the ovarian hormone estrogen. This paper supports our
previous observation that UG produced biological responses
suggestive of estrogen antagonism (7,8,15). UG decreased
mean uterine weight in intact mice and increased the length
of the estrous cycle. Since uterine weight is primarily under
the control of estrogen (30) and passage through the estrous
cycle is dependent on estrogen and progesterone (30), these
effects suggest that UG has the characteristics of an antiestrogen. The return of relative uterine and ovary weight and
the length of the estrous cycle toward normal values upon
withdrawal of UG further support our hypothesis that UG has
estrogen antagonistic effects. Furthermore, these data show
that modulations in relative organ weight and length of the
estrous cycle are dependent upon continuous UG exposure.
The mechanism of UG-induced estrogen antagonism is
unknown. However, we previously demonstrated that UG does
not alter serum 17-β estradiol levels in cycling mice (8), does
not competitively bind to uterine estrogen receptors in Ovex
mice after acute exposure or intact mice after subchronic
exposure (8,15), does not down-regulate uterine estrogen
receptors (8), does not change uterine peroxidase activity in
mature mice after subchronic exposure or after acute exposure
in immature mice (7,15), does not alter uterine weight in Ovex
mice after various acute treatment regimens (7), does not
decrease estrogen-induced increases in uterine weight (7,8),
does not block 3H 17-β estradiol uterine uptake or 3H in serum
(7), and does not alter uterus or ovary histology (7,8,15).
Thus, UG-induced anti-estrogenic effects concomitant with
hepatocarcinogenesis are apparently not mediated through the
estrogen receptor.
Uterine weight and passage through the estrous cycle are
primarily under the control of estrogen (30). Since estrogen is
principally secreted by the ovaries, the decrease in uterine
weight and increase in length of the estrous cycle may be
secondary to ovarian toxicity. The decrease in ovary weight
after UG exposure is consistent with ovarian toxicity. However,
there was no histological evidence of ovarian toxicity in UGexposed mice. Microscopic alterations in the ovary are difficult
to detect, especially if they are subtle. However, a lack of
ovarian toxicity is further supported by the fact that UG-
Tumor-promoting ability of unleaded gasoline
exposed mice continued to cycle through the various stages
of estrous, albeit at a slower rate. Since endocrine function is
under the control of a number of organ systems and feedback
mechanisms (31–34), UG may produce its anti-estrogenic
effects indirectly by modulating other parts of the endocrine
system.
Tumor promoters increase foci growth by altering gene
expression (35). In DEN-initiated female B6C3F1 mice, ingestion of estrogen decreases the size of hepatic foci and focal
LI while UG vapor increases the size of hepatic foci and focal
LI relative to DEN/control foci (6–8). Hepatic growth is under
the control of both positive and negative growth factors (36–
38). Over 95% of UG-promoted hepatic foci demonstrated
immunohistochemical downregulation of transforming growth
factor-beta1 (TGF-β1) and mannose-6-phosphate receptor
(M6PR) relative to normal hepatocytes, while 50% of estrogentreated foci elicited an upregulation of TGF-β1 (39). TGF-β1
is a negative regulator of hepatocyte growth that is secreted
in a latent form (40–42). M6PR facilitates proteolytic activation
of latent TGF-β1 to its mature active form (43). The differential
effect of UG on TGF-β1 and M6PR in focal hepatocytes
relative to non-focal hepatocytes may provide a selective
growth advantage to UG-promoted hepatic foci. Furthermore,
the increase of TGF-β1 in foci of estrogen-treated mice may
provide clues as to the mode by which ovaries normally inhibit
focal growth.
There are a number of short-term effects believed to be
involved in the hepatic tumor-promoting ability of chemicals.
Many hepatic tumor promoters produced hepatomegaly and a
transient increase in hepatocyte proliferation in the absence of
hepatotoxicity and induced hepatic PROD and EROD activity,
isozymes of the cytochrome P450 family (44–48). There is a
high correlation between the induction of hepatic PROD
activity and tumor promoting ability (18,49). EROD is a
P450 enzyme that metabolizes endogenous estrogen. Many
antiestrogens induce EROD activity which may contribute to
their tumor promoting properties by increasing hepatic estrogen
metabolism (50). In intact mice, UG increased liver size,
produced a transient increase in hepatocyte DNA synthesis,
and induced PROD and EROD activity (2,8,48). We previously
observed that 1 week of UG exposure also significantly
increased liver weight and hepatocyte proliferation in Ovex
B6C3F1 mice and to a degree similar to that produced by
UG in intact mice (personal observation). In this series of
experiments, subchronic UG exposure increased relative liver
weight and induced PROD and EROD activity to an approximately equal extent in both intact and Ovex mice. However,
UG had very little if any tumor promoting activity in Ovex
mouse liver. Therefore, hepatomegaly, transient increase in
DNA synthesis, and P450 induction although they may be
related to the tumor-promoting ability of some chemicals are
not specific markers for agents that have hepatic tumorpromoting activity.
Formulations of UG vary over time and with manufacturers.
The formulation of PS-6 UG, developed in the 1970s was
used in the chronic bioassay and the earlier tumor promotion
experiment (2,7,8). Our study, for the first time, clearly showed
that a newer formulation of UG 91-01 produced in 1991 also
had tumor-promoting ability. The absolute and relative increase
in the volume fraction of liver occupied by foci (4.17% and
3.5-fold, respectively) as compared to controls after 4 months
of 91-01 UG exposure in this study was strikingly comparable
to earlier data using PS-6 UG (4.31% and 3.8-fold, respectively)
and confirms the tumor-promoting ability of UG in intact
B6C3F1 mouse liver (8). Thus, even though 91-01 UG has a
slightly greater percentage of aromatics and olefins and a
lower percentage of saturated hydrocarbons than PS-6 UG,
the two formulations of UG have similar hepatic tumor
promoting ability. The hepatic DNA-synthesis and cytochrome
P450 inducing ability of UG resides in the high boiling point
fractions of UG (51). However, the specific component or
components of UG which are responsible for the hepatic
tumor-promoting ability of UG are unknown.
In conclusion, we demonstrated that in DEN-initiated Ovex
mice UG exhibited greatly attenuated hepatic tumor promoting
activity. The importance of the ovaries with regard to the
tumor-promoting activity of UG probably resides with the
mouse hepatocarcinogenic inhibitor estrogen, and is consistent
with our hypothesis that the tumor promoting ability of UG is
secondary to its interaction with estrogen. In addition to UG,
there are other hepatic carcinogens such as methylene chloride
(52,53), hexachloroethane (54), 1,1,2-trichloroethane (55),
chlordane (56,57), and MTBE (58) that are generally nongenotoxic in short-term in vitro tests of genetic toxicity,
increased the incidence of liver cancer in female mice, and also
produced uterine alterations suggestive of estrogen antagonism.
Therefore, understanding the mechanisms of UG-induced liver
tumorigenesis may have implications for a number of other
mouse hepatocarcinogens.
Acknowledgements
We gratefully acknowledge Paul Ross and Ardie James for co-ordinating
animal care and inhalation exposures, respectively, and Mike Judge, Carol
Bobbitt, Kathy Bragg, Steve Butler, Richard Masney, Mary Morris, Corrie
Dunn, Otis Lyght, Delorise Williams, Kay Roberts and Carl Parkinson for
technical assistance. We also thank Dr Derek Janszen for statistical advice,
Dr Barbara Kuyper for editorial comments, and Dr Julian Preston, Dr Leslie
Recio, Dr Ronny Fransson-Steen and Dr Russell Cattley for review of the
manuscript. This work was supported in part by a grant from the American
Petroleum Institute and NIEHS ES-05641-01 from the National Institute of
Environmental Health Services.
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Received on November 1, 1996; accepted on January 8, 1997
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