TOMCOLOGICAL SCIENCES 44, 1 4 - 2 1 (1998)
ARTICLE NO. TX982474
HIGHLIGHT
Dose-Dependent Metabolism of Benzene in Hamsters, Rats, and Mice
James M. Mathews,* Amy S. Etheridge,* and H. B. Matthewst
* Center for Bioorganic Chemistry, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709;
and ^National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Received December 23, 1997; accepted April 10, 1998
and Kalf, 1994). As a result of the lack of appropriate industrial
hygiene, benzene has been observed to induce bone marrow
depression leading to aplastic anemia (Selling, 1916; Snyder
and Kocsis, 1975; Snyder and Kalf, 1994) and acute myelogenous leukemia in persons exposed occupationally (Infante et
al, Wll; Snyder and Kalf, 1994). The source of these toxicities has been extensively investigated in animals and is generally attributed to metabolites of benzene rather than the
parent compound (Guy et al, 1991; Snyder and Kalf, 1994;
Snyder and Hedli, 1996). In rats and mice, absorbed benzene is
exhaled unchanged and converted to benzene oxide (Lovem et
al, 1997), phenol, hydroquinone, catechol, trans,trans-muconic acid, phenylmercapturic acid, 1,2,4-trihydroxybenzene,
and the respective glucuronide or sulfate conjugates of the
phenolic metabolites (Sabourin et al., 1988a). The precursor of
trans.trans-muconic acid, muconaldehyde, likely in combination with hydroquinone and/or other hydroxylated metabolites
is thought to account for benzene toxicity (Guy et al., 1991;
Snyder and Hedli, 1996).
Exposure to benzene is still an occupational hazard in developing countries where it is used extensively as a solvent for
latex glues used in the manufacture of shoes and in other
manufacturing processes (Bechtold et al, 1992). Occupational
exposure in more developed countries has been greatly decreased by the implementation of modern industrial hygiene
(Runion and Scott, 1985). In the United States and Europe
exposure of the general public is primarily through mainstream
cigarette smoke and, for nonsmokers, auto exhausts and gasoline vapor emissions (Lebret et al., 1986; Krause et al, 1987;
Wallace etal, 1987; Wallace, 1996). A common characteristic
of exposures in the more developed countries is that they
involve far lower doses than those known to induce toxicity.
Further, most exposures encountered by humans, occupationally or environmentally, are currently significantly lower than
those used to characterize benzene metabolism and toxicity in
laboratory animals (Runion and Scott, 1985; NTP, 1986; Sabourin et al., 1988a; Huff et al., 1989; Wallace, 1989; Wallace
et al, 1989; Snyder and Hedli, 1996). Thus, it is necessary to
base assessments of human health risks associated with current
Dose-Dependent Metabolism of Benzene in Hamsters, Rats, and
Mice. Mathews, J. M., Etheridge, A. S., and Matthews, H. B.
(1998). ToxicoL ScL 44, 14-21.
The disposition of oral doses of [14C]benzene was investigated
using a range of doses that included lower levels (0.02 and 0.1
mg/kg) than have been studied previously in rat, mouse, and in
hamster, a species which has not been previously examined for its
capacity to metabolize benzene. Saturation of metabolism of benzene was apparent as the dose increased, and a considerable
percentage of the highest doses (100 mg/kg) was exhaled unchanged. Most of the remainder of the radioactivity was excreted
as metabolites in urine, and significant metabolite-specific changes
occurred as a function of dose and species. Phenyl sulfate was the
predominant metabolite in rat urine at all dose levels (64-73% of
urinary radioactivity), followed by prephenylmercapturic acid
(10-11%). Phenyl sulfate (24-32%) and hydroquinone glucuronide (27-29%) were the predominant metabolites formed by mice.
Mice produced considerably more muconic acid (15%), which is
derived from the toxic metabolite muconaldehyde, than did rats
(7%) at a dose of 0.1 mg/kg. Unlike both rats and mice, hydroquinone glucuronide (24-29%) and muconic acid (19-31%) were the
primary urinary metabolites formed by hamsters. Two metabolites
not previously detected in the urine of rats or mice after single
doses, 1,2,4-trihydroxybenzene and catechol sulfate, were found in
hamster urine. These data indicate that hamsters metabolize benzene to more highly oxidized, toxic products than do rats or
mice.
C 1998 Society of Toxicology.
Benzene has been widely used for over 100 years and
continues to be used extensively as a solvent and synthetic
intermediate. Approximately 16 billion pounds of benzene
were used in the United States in 1996 (Chem. Eng. News,
1996). Benzene is also present in gasoline at 1-2%. Reports of
benzene toxicity to humans exposed occupationally were first
reported 100 years ago (Snyder and Kalf, 1994). However, the
extensive use of benzene continued to be quite casual and
largely unregulated until relatively recently (Parke, 19%)
when human toxicities were confirmed through epidemiology
studies and further investigated in studies with animals (Snyder
1096-6080/98 S25.00
Copyright O 1998 by the Society of Toxicology.
AU rights of reproduction in any form reserved.
14
15
METABOLISM OF BENZENE rN MAMMALS
benzene exposure on results from a range of exposures for
which metabolism and toxicity can only be assumed to be
linear. It is not yet possible to characterize the metabolism of
benzene at the low ppm levels to which most of the population
is exposed. However, with the objective of providing better
data for risk assessment, the present study was designed to
determine the fate of benzene in rats and mice at significantly
lower doses than those addressed previously. This work also
includes some of the first studies of the fate of benzene across
a range of doses in a third species, the hamster, and its
evaluation as an important new animal model for studying the
hematopoietic toxicity induced by benzene metabolites working alone or in concert.
METHODS
Animals. F344 rats and B6C3F1 mice were purchased from Charles River
Laboratories, Inc. (Raleigh, NC) and were selected for this work to be consistent with the studies of me National Toxicology Program. LVG hamsters
were purchased from Charles River Laboratories (Kingston, NJ). At dosing,
the age and weight ranges for rats were 65-73 days and 188-233 g, for mice
were 81 days and 25-27 g, and for hamsters were 52-55 days and 100-122 g.
All animals were males and were quarantined at least 1 week before they were
used in a study. Animals were fed Certified Purina Rodent Chow 5002 and
were furnished tap water ad libitum. They were transferred from standard
polycarbonate cages to individual glass metabolism chambers the day before
they were used in an experiment These chambers provided for separate
collection of urine and feces and collection of breath.
Test materials. [U-l4C]Benzene (117 mCi/mmol) was prepared by Chemsyn Science Laboratories (Lenexa, KS) and was found to be ca. 99% pure by
HPLC analysis. The chromatographic system consisted of a Zorbax ODS
analytical column (4.6 x 250 mm, 5 jim) with an isocratic mobile phase of
60% ethanol and 40% water at a flow rate of 1 ml/min. Column effluent was
monitored with a /3-RAM radioactivity detector equipped with a 0.5-ml solid
scintillate flow cell. Non-radiolabeled benzene was supplied by Aldrich Chemical Company, Inc. (Milwaukee, WT).
Metabolite standards. Phenyl mercapturate was synthesized by the
method of Behringer and Fackler (1949). Catechol (99+%), hydroquinone
(99+%), 1,4-benzoquinone (98%), muconic acid (98%), and phenol
(99+%) were obtained from Aldrich Chemical Company (Milwaukee, WI).
1,2,4-Benzenetriol (98%) was obtained from Pfaltz and Bauer, Inc. (Waterbury, CT). Prephenyl mercapturate was identified and quantitated following
acid-catalyzed conversion to phenyl mercapturate as described by Sabourin
et al. (1988b).
Preparation and administration of dose. Each dose for rats, mice and
hamsters contained 12-26, 4 - 2 1 , and 3-22 yJZ\ radiolabel, respectively. Additionally, all dose formulations contained an appropriate amount of unlabeled
benzene and corn oil in a single dose volume of 5 ml/kg (rats and hamsters) or
25 ml/kg (mice). Doses were administered by intragastric gavage.
Collection and analysis of biological samples. Urine and feces of rats,
mice, and hamsters were collected separately into round-bottom flasks cooled
with dry ice. Urine and feces were collected at 4, 8 (urine only), 24, and 48 h
and were stored in the dark at -20°C until analyzed. Radiolabeled components
in breath were trapped in cryogenic traps containing ethanol as previously
described (Mathews et aL, 1991). Radiolabeled metabolites) present in the
traps was analyzed by HPLC, using the method described above for the
analysis of the radiochemical purity of the test material.
At the end of each experiment, rats and hamsters were anesthetized with an
im injection of 60 mg/kg ketamine and 8.6 mg/kg xylazine. Mice were
anesthetized with a 180 mg/kg im injection of sodium pentobarbital. Rats and
hamsters were euthanized by an intracardiac injection of sodium pentobarbital
(300 mg/kg) and mice were euthanized by cervical dislocation.
Aliquots of dose formulation, urine, and trapping solution from the breath
traps were added directly to vials containing scintillation cocktail (Ultima
Gold, Packard Instrument Company). Samples of feces (0.1-0.3 g) were
digested in Soluene-350 (2 ml). After digestion, samples requiring bleaching
were decolorized with perchloric acid/hydrogen peroxide prior to addition of
scintillation cocktail (Ultima Gold, Packard Instrument Company).
Urinary metabolite profiles were determined for rats, mice, and hamsters by
HPLC analysis using a modification of the method of Sabourin et al. (1988b).
Rat and hamster urine samples were prepared for each animal at each dose
level by combining 10% of each urine sample collected 0-24 h postdosing.
Similarly, composite urine samples from individual mice were prepared for
each animal by combining urine collections made 0-24 h postdosing. Pooled
urine samples were then filtered through a 0.45-^un Millex-HV filter and
analyzed by HPLC. The chromatographic system consisted of a DuPont
Zorbax ODS analytical column. Urinary metabolites were eluted using a linear
gradient, changing from 10 to 90% methanol in 35 mM aqueous tetrabutylammonium hydrogen sulfate (Sigma Chemical Co., St. Louis, MO) over a 35-min
period at a flow rate of 1 ml/min. Column effluent was monitored using both
UV absorbance at 270 nm (Applied Biosystems 759a) and a 0-RAM flowthrough radioactivity detector equipped with a 0.5-ml solid scintillate flow cell.
Following the injection of each urine sample, the column effluent was collected
in fractions directly into scintillation cocktail. The radioactivity eluting in each
fraction was measured by liquid scintillation spectrometry. Urinary metabolites
were identified by coelution with metabolite standards.
In order to determine which metabolites were present as sulfate or glucuronide conjugates, pooled urine samples were incubated with either sulfatase and
0-glucuronidase (Sigma Chemical Co.). Sulfatase incubations included 100 /xl
of urine combined with Trizma buffer (pH 7.6, 100 fi\) and sulfatase (100 /il,
Sigma; prepared from Aerobacter aerogenes, 18.9 U/ml). The incubations
were then allowed to react at 37°C for 16 h. /3-Glucuronidase reactions were
initiated by the addition of 200 fi\ of urine to purified enzyme (Sigma; prepared
from Escherichia coli, 1000 U) and were allowed to incubate at 37°C for 1 h.
Incubation samples were then analyzed by HPLC and these profiles were
compared with HPLC profiles of the urine samples prior to enzyme incubation.
The activity of cytochrome P450 2E1 in hepatic microsomes prepared from
rats, mice, and hamsters was determined by measuring the rate of the hydroxylation of p-nitrophenol as previously described (Mathews et aL, 19%).
Statistical analysis. Values were compared using ANOVA followed by a
one-sided Dunnett's test Statistically significant differences were determined
at the a = 0.05 level.
RESULTS
Excretion of benzene-derived radioactivity following oral
administration of a range of [14C]benzene doses to rats is
shown in Table 1. Following a dose of 0.02 mg/kg, 99% of the
radiolabel was recovered in urine, with <60% being excreted
within the first 8 h and <99% after 24 h. Total elimination
continued to be rapid as the dose was increased and accounted
for greater than 90% of the dose in the first 24 h in every case.
The pattern of excretion in urine and breath was dose dependent. Excretion in urine decreased from nearly 100% to less
than 50% as the dose increased from 0.1 to 100 mg/kg. This
was apparently due to the increasing the percent of dose
exhaled in breath as volatiles from about 2% at a dose of 0.1
mg/kg to 50% at a dose of 100 mg/kg. Elimination as CO 2
(data not shown) or in feces never accounted for more than 2%
of the dose. However, in all cases greater than 90% of the dose
16
MATHEWS, ETHERIDGE, AND MATTHEWS
TABLE 1
Disposition of Radioactivity 48 h after Oral Administration of [MC]Benzene to Male F-344 Rats (N = 4)
Cumulative percentage of dose in:
End of
Dose (mg/kg)
0.02
0.1
0.5
10
100
collection
period (h)
4
8
24
48
4
8
24
48
4
8
24
48
4
8
24
48
4
8
24
48
Urine
37.3
60.2
96.1
99.3
14.6
63.1
95.4
99.3
22.8
42.1
87.8
95.4
40.5
68.2
90.0
93.2
2.2
18.1
45.5
47.5
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
12.9
18.1
2.3
0.7
25.3
13.5
1.9
1.0
17.2
13.3
3.0
2.2
9.8
7.2
3.2
3.8
2.3
4.4
2.3
2.9
Breath
1.8 ± 0.6
2.2 ± 0.8
2.3 ± 0.7
NC
NC
NC
0.5 ±0.1
NC
1.9 ± 1.1
2.3 ± 1.3
2.6 ± 1.3
NC
8.6 ± 2.4
9.2 ± 2.6
9.4 ± 2.7
NC
37.8 ± 4.7
48.6 ± 4.4
50.5 ± 4.6
NC
Feces
0.1
0.2
1.9
2.4
±0.2
± 0.2
±0.5
± 0.5
1.5
2.0
2.0
0.0
0.0
1.5
1.8
0.0
0.3
1.4
1.6
0.0
0.0
0.3
0.4
±0.1
± 0.2
± 0.2
± 0.0
± 0.0
±0.2
±0.2
± 0.0
± 0.5
±0.4
±0.4
± 0.0
± 0.0
± 0.1
± 0.1
Total
38.3
62.6
100.3
103.9
15.0
65.1
97.5
101.9
24.8
44.5
91.9
99.9
49.1
77.8
100.9
104.3
40.1
66.8
96.4
98.5
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
13.0
17.6
2.1
0.5
25.0
13.6
2.0
0.9
17.3
12.7
3.3
2.3
11.7
7.7
2.5
3.3
4.0
5.9
2.8
3.2
* Significantly different from single 10 mg/kg dose.
" NC, not collected.
was eliminated in the first 24 h after administration. A separate
experiment in which benzene was administered intraperitoneally demonstrated that volatile radioactivity recovered from
breath originated from absorbed, rather than refluxed, benzene
(data not shown).
Virtually all (96%) of the benzene-derived radioactivity
eliminated in expired breath was present as the parent compound (data not shown). The identities of metabolites eliminated in urine were confirmed by coelution on HPLC with
known standards and were consistent with those described
previously (Sabourin et al., 1988a). Profiles of metabolites
present in urine 24 h after oral doses of benzene ranging
from 0.02 to 100 mg/kg are shown in Table 2. The relative
amounts of benzene metabolites present at 48 h were also
determined for each species studied. However, since the
amount excreted in the second 24-h period was minimal and
the pattern of metabolites did not vary significantly from
those seen in the first 24 h, these data are not presented.
Phenyl sulfate was the predominant metabolite at all dose
levels, comprising 64-73% of total urinary radioactivity,
and tended to increase slightly with dose. Phenylmercapturic acid also increased slightly with increasing dose. Prephenylmercapturic acid was the second most abundant metabolite and comprised 9 - 1 1 % of the urinary metabolites.
The relative amounts of radioactivity present as prephenylmercapturic acid and hydroquinone varied little with
dose. In contrast, the relative amount of muconic acid pro-
duced was greatest at the lowest doses to the extent that
muconic acid was the third most prevalent metabolite at the
three lowest doses.
Excretion of radioactivity during the first 48 h following oral
administration of [14C]benzene doses of 0.1 and 100 mg/kg to
mice is shown in Table 3. Elimination was rapid and primarily
in urine. Mice excreted considerably less of the dose unrnetabolized, and 2 and 23% of the 0.1 and 100 mg/kg doses,
respectively, were recovered as volatile equivalents in breath.
Most of the dose expired as volatiles was recovered in the first
4 h postdosing. Less than 1% of the dose was excreted as
carbon dioxide (data not shown). Urinary and fecal excretion
of the dose accounted for 95% of the low dose and 74% of the
high dose. Unlike rats, a significant portion of both doses was
recovered in feces. However, it is very common for mouse
fecal collections to be contaminated with urine, and it is
speculated that the data do not reflect a true difference in the
routes of excretion between the species.
Profiles of urinary metabolites eliminated by mice in the
first 24 h after oral doses of 0.1 and 100 mg/kg are shown in
Table 4. Unlike the rat, about as much hydroquinone glucuronide as phenyl sulfate was produced at both doses, and
together these two metabolites accounted for over half of the
urinary radioactivity. Muconic acid accounted for a larger
portion of the urinary radioactivity (12-15%) than observed
in rats (2-7%), but was less affected by dose and accounted
for only a slightly smaller relative amount of the high dose.
17
METABOLISM OF BENZENE IN MAMMALS
TABLE 2
Profile of Metabolites" Appearing in the Urine of Rats Administered [uC]Benzene
<*> of Urinary radioactivity appearing as:
% of Dose
Urine sample6
recovered in
urine in 24 h
UNK
HQG
HQ
MUC
PPMA
Phenol
PMA
HQS
PS
PG
Total
95.82
2.75
1.51
0.16
3.96
0.51
0.31
0.04
7.45
0.79
9.29
0.51
2.58
0.32
1.65
0.08
<0.05
68.59
0.54
1.02
0.08
96.38
0.36
96.40
1.17
3.44
0.83
6.13
0.92
0.53
0.07
6.06
1.51
10.26
1.43
5.71
0.42
0.89
0.01
<0.05
63.89
2.84
1.33
0.19
98.55
1.08
89.30
1.94
2.01
0.65
4.26
0.06
0.37
0.11
4.85
0.96
10.61
0.66
3.56
0.29
1.97
0.32
<0.05
69.33
1.41
1.48
0.14
98.86
0.54
89.44
3.68
1.21
0.30
3.77
0.52
0.54
0.07
3.62
1.28
11.24
1.66
4.20
0.16
1.52
0.23
<0.05
70.32
0.49
1.67
0.20
98.09
1.09
44.80
2.26
1.00
0.23
2.19
0.37
0.33
0.15
1.77
1.00
10.99
0.54
3.01
0.56
2.82
0.87
<0.05
73.42
1.41
1.57
0.57
97.11
0.84
0.02 mg/kg
Mean
SD
0.1 mg/kg
Mean
SD
0.5 mg/kg
Mean
SD
10 mg/kg
Mean
SD
100 mg/kg
Mean
SD
" Metabolites: UNK, unknown; HQG, hydroquinone glucuronide; HQ, hydroquinone; MUC, muconic acid; PPMA, prephenylmercapturic acid; PMA,
phenylmercapturic acid; HGS, hydroquinone sulfate; PS, phenyl sulfate; PG, Phenyl glucuronide.
* 0-24 h pooled urine collection.
Phenyl glucuronide accounted for a greater portion of the
dose than seen in the rat and the relative amounts increased
with dose, as did the percentage of urinary metabolites
present as phenyl sulfate and phenyl glucuronide. The relative amounts of most other metabolites were minor and
apparently minimally affected by dose.
In addition to the work done with rats and mice, the fate
of benzene in a third species was investigated in order to
determine if results seen for rats and mice are representative
of other mammalian models. In this case, the species of
choice was the hamster, which has not been previously
examined for its capacity to metabolize benzene, but is
known to actively metabolize numerous other xenobiotics.
Excretion of radioactivity during the first 48 h following
oral administration of a range of 14C-labeled benzene doses
to hamsters is shown in Table 5. Total elimination by
hamsters was less rapid than observed in rats and more rapid
than mice, with elimination in urine the predominant means
of excretion for each species. As seen for both rats and mice,
elimination as volatiles in breath by hamsters was minimal
at the low doses and significant at the high dose. Total
elimination as volatiles in breath by hamsters was most
similar to that observed for mice. Elimination as CO 2 ( ^ 1 % ,
data not shown) or in feces by hamsters was minimal at all
doses, as was the case with rats and mice. Minimal elimination of benzene-derived radioactivity in feces by hamsters
receiving the high dose was most similar to that observed
for rats.
TABLE 3
Disposition of Radioactivity 48 h after Oral Administration of [l4C]Benzene to Male B6CF1 Mice (TV = 4)
Cumulative percentage of dose in:
Dose (mg/kg)
0.1
100
' NC, not collected.
End of collection
period (h)
4
8
24
48
4
8
24
48
Urine
0.02
0.02
55.8
76.1
0.0
1.9
16.3
43.1
±
±
±
±
±
±
±
±
0.0
0.0
10.3
8.7
0.0
3.3
14.5
21.9
Breath
1.3 ± 0.9
1.4 ± 1.0
1.6 ± 1.0
NC
20.1 ± 3.7
21.6 + 4.4
22.6 ± 4.6
NC
Feces
0.01
1.0
5.5
18.8
0.0
0.3
13.19
31.1
± 0.0
± 0.5
± 1.8
± 7.4
± 0.0
± 0.4
± 18.2
±23.5
Total
1.7
3.0
63.4
97.0
20.3
24.0
52.7
97.5
±
±
±
±
±
±
±
±
1.0
0.6
9.7
3.3
3.7
2.7
3.6
1.5
18
MATHEWS, ETHERIDGE, AND MATTHEWS
TABLE 4
Profile of Metabolites" Appearing in the Urine of Mice Administered [14C]Benzene
% of Urinary radioactivity appearing as:
% of Dose
Urine
sample*
0.1 mg/kg
Mean
SD
100 mg/kg
Mean
SD
recovered in
urine in 24 h
UNK
HQG
HQ
MUC
PPMA
Phenol
PMA
HQS
PS
PG
Total
55.80
10.31
7.39
0.65
31.97
1.31
1.40
0.17
15.32
1.93
2.03
0.28
2.95
0.29
3.71
1.31
2.84
0.34
23.69
0.61
3.08
0.09
94.37
0.19
24.82
5.43
5.24
0.89
26.49
1.66
1.22
0.20
11.72
1.67
2.25
1.36
2.61
0.29
2.66
0.31
2.29
0.41
31.68
0.46
7.88
1.28
94.03
0.70
" Metabolites: UNK, unknown; HQG, hydroquinone glucuronide; HQ, hydroquinone; MUC, muconic acid; PPMA, prephenylmercapturic acid; Phenol,
phenol; PMA, phenylmercapturic acid; HQS, hydroquinone sutfate; PS, phenylsulfate; PG, phenyl glucuronide.
* 0-24 h pooled urine collection.
The major species differences in the fate of benzene in rats,
mice, and hamsters were seen in the profiles of metabolites
excreted in urine in the first 24 h following dosing (Table 6).
Unlike both rats and mice, phenyl sulfate was not the predominant urinary metabolite found in hamsters. At all doses, hydroquinone glucuronide and muconic acid were the primary
urinary metabolites excreted by hamsters. The amount of hydroquinone glucuronide excreted was similar to that observed
for mice and the relative amounts decreased only slightly as the
dose increased. The relative amount of muconic acid excreted
by hamsters was 4 to 10 times that excreted by rats and twice
that excreted by mice. Muconic acid excretion by hamsters
decreased by about a third when the dose was increased from
0.02 to 100 mg/kg. Two metabolites not detected in single dose
studies with rats and mice, 1,2,4-trihydroxybenzene and catechol sulfate, were found in small amounts in hamster urine.
The presence of these metabolites suggests that hamsters metabolize benzene to more highly oxidized products. As the
progressive oxidation of benzene is mediated by CYP2E1
(Valentine et al, 1996), the activity of that isozyme was
measured in these studies. Rates for the CYP2E1-mediated
hydroxylation of p-nitrophenol by hepatic microsomes prepared from rat, hamster, and mouse were 0.97 ± 0.21, 4.62 ±
0.85, and 4.74 ± 0.51 nmol/mg-min, respectively.
DISCUSSION
As described in a recent review by Snyder and Hedli (1996),
the metabolism of benzene has been very well characterized.
Further, the metabolic pathways and resulting metabolites are
qualitatively similar for each species studied, though there may
be significant quantitative differences (Sabourin et al., 1988a;
Medinsky et al, 1989; Snyder and Hedli, 1996; Henderson,
1996). The metabolism of benzene may result in either activation or deactivation of its toxicity; thus, it is these quantitative
differences that are thought to account for the varying sensi-
TABLE 5
Disposition of Radioactivity 48 h after Oral Administration of [14C]Benzene to Male LVG Hamsters (N = 4)
Cumulative percentage of dose recovered in:
Dose (mg/kg)
0.02
0.1
100
End of collection
period (h)
4
8
24
48
4
8
24
48
4
8
24
48
" NC, not collected.
Urine
0.0
33.4
68.5
81.7
20.5
51.3
80.8
89.3
3.0
25.1
51.2
60.2
±
±
±
±
±
±
±
±
+
+
±
+
0.0
24.7
7.7
7.8
24.4
9.9
7.1
4.4
6.1
9.1
3.8
3.0
Breath
1.4 ±0.6
1.7 + 0.5
2.0 ± 0.5
NC
1.6 + 0.2
1.8 + 0.2
2.0 ± 0.3
NC
24.5 + 1.7
27.7 + 2.1
29.0 ±2.1
NC
Feces
0.0
0.4
5.4
6.8
0.0
0.2
1.7
3.4
0.0
0.0
1.5
2.7
±
+
±
±
±
±
±
+
±
±
±
±
0.0
0.5
4.8
5.2
0.0
0.2
0.8
0.7
0.0
0.0
0.2
1.1
Total
1.7 :
36.9:
78.3 :
93.0:
22.4:
54.0:
85.5:
95.7:
27.7:
53.0:
82.1 :
92.3 :
: 0.7
25.9
3.5
2.3
24.3
: 9.8
7.4
4.5
5.5
10.1
: 3.8
3.2
19
METABOLISM OF BENZENE IN MAMMALS
TABLE 6
Profile of Metabolites" Appearing in the Urine of Hamsters Administered [14C]Benzene
%of
Urine
sample*
0.02 mg/kg
Mean
SD
0.1 mg/kg
Mean
SD
100 mg/kg
Mean
SD
%of Urinary radioactivity appearing as:
Dose
recovered
UNK
HQG
HQ
MUC
PPMA
Phenol
PMA
HQS
PS
PG
THB
cs
Total
65.78
6.61
3.63
0.91
27.92
1.18
0.95
0.30
30.54
1.21
2.19
0.94
7.05
0.58
3.51
0.79
2.18
0.89
9.76
0.43
6.26
0.63
0.20
0.35
1.75
0.20
95.93
1.72
78.11
5.86
3.84
1.03
29.18
2.73
2.04
0.25
26.98
2.17
4.44
1.70
8.68
1.09
2.94
1.51
0.74
0.28
8.47
2.02
6.39
0.14
1.83
0.36
NDC
ND
95.54
0.10
51.14
4.61
3.08
0.44
24.13
2.43
0.90
0.69
18.99
0.80
5.02
0.83
8.74
0.37
2.24
0.57
1.69
0.72
12.62
2.04
14.95
0.24
2.59
0.71
1.57
0.06
96.51
1.65
° Metabolites: UNK, unknown; HQG, hydroquinone glucuronide; HQ, hydroquinone; MUC, muconic acid; PPMA, prcphenylmercapturic acid; Phenol,
phenol; PMA, phenylmercapturic acid; HQS, hydroquinone sulfate; PS, pbenyl sulfate; PG, phenyl glucuronide; THB, trihydroxybenzene; CS, Catechol sulfate.
* 0-24 h pooled urine collection.
c
ND, not detected.
tivity of different species to benzene intoxication and carcinogenesis (Henderson et al, 1989; Snyder and Kalf, 1994; Henderson, 1996). The primary source of variation among species
is most probably the varying amounts of the hepatic enzyme
thought to account for most benzene metabolism, CYP2E1
(Johansson and Ingelman-Sundberg, 1988; Koop et al., 1989;
Valentine et al, 1996). Since studies of chemical toxicity are
designed to elicit an effect, it is likely that many of the studies
on which knowledge of benzene toxicity is based may have
used doses that saturated the capacity of the test species to
metabolize this xenobiotic. This fact does not in any way
negate the findings of these studies, but the resulting data may
not be optimal for the support of assessments of human health
risks associated with exposure to low levels of benzene. The
objective of the present study was to provide data describing
the fate of benzene when administered at lower does than used
previously in order to establish a linear range of metabolism.
This study also presented an opportunity to examine benzene
metabolism in the hamster, a species known for its capacity to
metabolize many xenobiotics.
As demonstrated by numerous previous studies and by the
number of metabolites isolated in this study, the metabolism of
benzene is quite complex. Metabolites implicated in benzene
toxicity tend to be minor urinary metabolites in most species.
Phenol sulfate, the major metabolite in most species, and other
conjugated metabolites are thought to be relatively nontoxic,
though they may represent metabolism through a toxic intermediate. When administered as individual compounds, minor
metabolites thought to account for toxicity, e.g., phenol, hydroquinone, or muconaldehyde, the toxic precursor of muconic
acid, fail to elicit the magnitude of toxicity observed with
benzene (Cox, 1991; Guy et al, 1991). Thus, it is thought that
the toxicity of benzene can be attributed to a combination of
metabolites that act additively or synergistically (Eastmond et
al, 1987; Barale et al., 1990; Cox, 1991; Guy et al, 1991;
Snyder and Hedli, 1996). These quantitative differences in the
rates and types of metabolites formed have been speculated to
account for the varying sensitivity of rats and mice to benzene
toxicity and carcinogenicity (Henderson et al, 1989; Henderson, 1996). However, as with toxicity studies, most of the
studies of benzene metabolism on which comparative estimates
are based used doses that may have saturated the metabolic
capacity of the test species. The present study endeavors to
address this deficiency in the literature.
Results of the present study demonstrate that benzene was
readily absorbed and then rapidly metabolized and eliminated
primarily in urine by each species studied. However, the routes
of elimination were somewhat altered by dose. At the high
dose of 100 mg/kg a significant portion of the dose was
eliminated in breath. Since benzene metabolites are thought to
account for its toxicity, benzene eliminated in breath is, in
effect, biologically unavailable. Elimination of a dose of 100
mg/kg in breath ranged from 22% in mice to 50% in the rats.
This observation confirmed a previous observation that a high
dose is metabolized to a greater extent in the mouse (Henderson, 1996). Elimination of the high dose of benzene in breath
by hamsters was more similar to that seen in mice and correlates with the higher CYP2E1 activities measured in the two
species. Only 1% of the dose was eliminated in breath by either
species at a dose of 0.1 mg/kg or lower. Thus, as opposed to the
high dose, essentially all of a low dose of benzene was absorbed and metabolized in each species studied.
A number of species- and dose-dependent variations were
observed for benzene metabolites excreted in urine. Of
particular interest for the rat was the fact that the relative
amount of muconic acid, the oxidation product of the toxic
metabolite muconaldehyde, increased by approximately
fourfold as the dose decreased from 100 to 0.02 mg/kg. As
reported previously (Sabourin et al, 1988a, 1989), mice
excreted significantly more muconic acid than rats. Mice
20
MATHEWS, ETHERIDGE, AND MATTHEWS
also formed 7 to 10 times as much hydroquinone as rats.
There were some quantitative differences, however, between the findings of Sabourin et al. (1989) and the present
work in the amounts of muconic acid and hydroquinone
conjugates detected in urine, although in each case higher
proportions of the doses were converted to these metabolites
as the dose level decreased. In particular, Sabourin et al.
(1989) found 2 to 3 times more muconic acid (as percentage of dose) in the urine of rats administered a 1 mg/kg
oral dose than found in the present work after a 0.5 mg/kg
dose. Additionally, while in both studies about 4% of the
dose was converted to hydroquinone conjugates, Sabourin et
al. found the bulk of this material to be present as the sulfate
rather than glucuronide conjugate. In the present study, the
glucuronide was the predominate hydroquinone conjugate,
with 0.05% of the urinary radioactivity detected as the
sulfate conjugate following the 0.5 mg/kg dose. The relative
amounts of these and other potentially toxic metabolites
excreted by mice were more constant across the range of
doses studied than observed in rats and hamsters.
This is the first report that hamsters metabolize a significantly greater portion of a dose of benzene to muconic acid
than do either rats or mice. The relative amounts of muconic
acid eliminated were also dose dependent, accounting for approximately 19% of the high dose versus 30% of the low dose
excreted in urine. Hamsters also metabolized as much of each
benzene dose to hydroquinone as did mice and, like mice,
excreted it primarily as the glucuronide conjugate. Additionally, hamsters, but not rats or mice, produced detectable
amounts of trihydroxybenzene and catechol sulfate. These observations indicate that if muconaldehyde, hydroquinone, catechol, and trihydroxybenzene are, as speculated (Eastmond et
al., 1987; Barale et al., 1990; Cox, 1991; Guy et al., 1991;
Snyder and Hedli, 1996), among the most toxic metabolites of
benzene and act synergistically, then the hamster should be one
of the most sensitive animal models for studies of benzene
toxicity.
In summary, data presented here support the differences
previously reported for benzene metabolism and elimination by
rats and mice (Sabourin et al., 1988a, 1989). These data also
illustrate that benzene metabolism through oxidative pathways
leading to toxic metabolites or intermediates may be saturated
at relatively low doses in all three species studied. The relative
amounts of toxic metabolites formed appear to be more dose
dependent in rats and, to a lesser extent, in hamsters than in
mice. Thus, extrapolations of risk based on data obtained with
rats may be nonlinear above a single dose as low as 0.5 mg/kg.
Data obtained with hamsters indicate significantly greater metabolism through toxic intermediates than previously reported
for other species. In man, hepatic CYP2E1 varies at least
50-fold (Ronis et al., 1996), and the presence of high percentages (ca. 30%) of the measured metabolites as muconic acid
and hydroquinone in the urine of industrial workers exposed to
low levels of benzene has been linked with hematoxicity in
those individuals (Rothman et al, 1996). Thus, the hamster
may be an appropriate model for the most sensitive, high
CYP2E1 activity, humans, and it may serve as a model to
provide better estimates of the degree to which muconaldehyde
and other metabolites account for benzene toxicity and/or
carcinogenicity.
ACKNOWLEDGMENTS
This work was performed under National Institute of Environmental Health
Sciences Contract NO 1-ES-15329. The authors are grateful to Dr. Michele
Medinsky for critically reviewing the manuscript and to Ms. Sherry A. Tallent
for her assistance in its preparation.
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