Toxicologic Pathology
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Epididymal Sperm Granuloma Induced by Chronic Administration of 2-Methylimidazole in B6C3F1 Mice
Yoshiro Tani, Paul M. Foster, Robert C. Sills, Po C. Chan, Shyamal D. Peddada and Abraham Nyska
Toxicol Pathol 2005 33: 313
DOI: 10.1080/01926230590922866
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Toxicologic Pathology, 33:313–319, 2005
C by the Society of Toxicologic Pathology
Copyright ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1080/01926230590922866
Epididymal Sperm Granuloma Induced by Chronic Administration
of 2-Methylimidazole in B6C3F1 Mice
YOSHIRO TANI,1 PAUL M. FOSTER,2 ROBERT C. SILLS,1 PO C. CHAN,3 SHYAMAL D. PEDDADA,4 AND ABRAHAM NYSKA1
1
4
Laboratory of Experimental Pathology, 2 Laboratory of Molecular Toxicology, 3 Toxicology Operations Branch, and
Biostatistics Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park,
North Carolina 27709, USA
ABSTRACT
Two-year mouse and rat bioassay studies of 2-methylimidazole (2-MI) conducted by the National Toxicology Program revealed that epididymal
sperm granuloma(SG)s occurred only in male B6C3F1 mice in a dose-related manner. The present study characterized 2-MI-induced SGs in these
epididymides. Groups of 50 male B6C3F1 mice were fed diets containing 0, 625, 1250, or 2500 ppm 2-MI for 105 weeks; the doses were equivalent
to average daily doses of approximately 13, 40, or 130 mg/kg. Testes and epididymides were histopathologically reexamined. 2-Methylimidazole
increased the incidence of epididymal SGs (0%, 0%, 6%, 12%, respectively). Histologically, most of the SGs exhibited rupture of epididymal ducts
with focal aggregations of macrophages in interstitia. Lesions occurred in the proximal caput of the epididymis and/or efferent ducts, not in the corpus
and cauda. In the testis, incidences of germinal epithelial atrophy (GEA) increased dose-relatedly (2%, 8%, 16%, 28%, respectively). All mice with
epididymal SG developed testicular GEA. The grading scores of testicular GEA tended to be more severe in mice with SGs than those without. No
epididymal SG or testicular GEA was observed in 6-month-interim-sacrificed mice. The results imply that 2-year treatment of B6C3F1 mice with
2-MI can induce epididymal SGs, primarily followed by more severe testicular GEA. The potential mechanism of SG induction by 2-MI is discussed.
Keywords. 2-Methylimidazole; epididymis; sperm granuloma; testis; germinal epithelium; mice.
INTRODUCTION
The imidazole derivative, 2-methylimidazole (2-MI1 ;
C4 H6 N2 ; CAS No. 693-98-1, Figure 1), produced globally by
chemical companies, is widely used as a starting or intermediate material in several chemical synthetic processes, such as
the production of drugs, dyes, pigments, photographic chemicals, and corrosion inhibitors for metals (Chemical Economics Handbook, 1995). The derivative 2-Methylimidazole
is also used as an accelerator for polymerization cross-linking
and a catalytic curing agent for epoxy resins (Bogdal et al.,
2002). In addition to its industrial use, 2-MI is contained in
cigarette smoke (Moree-Testa et al., 1984) and occurs in paper products and as an undesirable by-product in several food
products, including caramel coloring, soy sauce, Worcestershire sauce, wine, ammoniated molasses, and caramelcolored syrups (Nishie et al., 1969). This chemical is watersoluble, absorbed swiftly and completely, distributed rapidly
and extensively to a variety of tissues, and eliminated quickly
in rats (Johnson et al., 2002). The National Toxicology Program (NTP) conducted 2-year bioassay and related toxicity
studies for 2-MI because of a lack of specific carcinogenicity
test data in the literature.
The exposure of F344/N rats and B6C3F1 mice of both
sexes to 2-MI for 2 years resulted in chemical-related lesions
in many organs including, but not limited to, the liver, thyroid
gland, and spleen in rats and the liver, thyroid gland, spleen,
bone marrow, kidney, epididymis, and testis in mice (National
Toxicology Program, 2005). In the epididymis, chronic active inflammatory lesions and sperm granulomas (SGs) were
observed in male mice, but not male rats, in a dose-related
manner. The definition of chronic active inflammation in
the original study included several characteristics appearing
singly or in combination: (1) mononuclear cell infiltration,
(2) engorgement of ducts with spermatozoa, (3) multinucleated germ cells in the lumen, (4) vacuolization and/or disruption of lining cells, (5) coagulated protein and/or cell debris,
and/or (6) fibrosis/edema. Chronic active inflammation was
commonly associated with SG.
Sperm granuloma, a reaction of the male genital tissues to
extravasated spermatozoa, may affect the testis, epididymis,
or vas deferens (Jones et al., 1997; Foley, 2001). During
spermatogenic development, spermatids and spermatozoa
appear in the seminiferous tubules after immune tolerance
has been established. Once spermatozoa are leaked into the
extravascular tissue following rupture of the male genital
tract, they may be recognized as foreign bodies and induce
an inflammatory reaction (McDonald, 2000; Lanning et al.,
2002).
Chemical-induced SGs have been reported mainly in rats.
In the present study, we analyzed the histopathological characteristics and localization of epididymal SGs induced in
B6C3F1 mice following chronic treatment with 2-MI. Testes
were also examined to determine if there were any pathological correlations between the testis and epididymis, as doserelated germinal epithelial atrophy (GEA) of the testis was
also recorded in the original study.
Address correspondence to: Abraham Nyska, Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, P. O.
Box 12233, Mail Drop B3-06, Research Triangle Park, North Carolina
27709, USA; e-mail: [email protected]
1
Abbreviations: GEA, germinal epithelial atrophy; H&E, hematoxylin
and eosin; 2-MI, 2-methylimidazole; NTP, National Toxicology Program;
SG, sperm granuloma.
313
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TOXICOLOGIC PATHOLOGY
TANI ET AL.
FIGURE 1.—Chemical structure of 2-methylimidazole.
MATERIALS AND METHODS
Chemicals
Bulk 2-MI (lot Nos. 08222CN and 04209TQ) was purchased from Aldrich Chemical Company (Milwaukee, WI)
and, after the two lots were combined, analyzed for purity
using a gas chromatograph. The purity of the bulk 2-MI was
99.5% or higher throughout the study. Dose formulations
of 2-MI, admixtures with powdered NTP-2000 diet (Zeigler
Brothers, Inc., Gardners, PA), were prepared and stored at
room temperature for up to 35 days. Preadministration dose
analysis was performed using high performance liquid chromatography.
Animals and Study Design
The original study was conducted by the NTP to evaluate
the chronic toxicity and carcinogenicity of 2-MI (National
Toxicology Program, 2005). Animal studies were conducted
under contract at Southern Research Institute (Birmingham,
AL). All animals were under the supervision of a veterinarian
and housed at a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care for
the duration of this study. Animal handling and husbandry
were conducted in accordance with NIH guidelines. Male
and female F344/N rats and B6C3F1 mice were obtained
from Taconic Farms, Inc. (Germantown, NY). Rats in the
chronic core study (total of 240 rats/sex) were randomly assigned to 1 of 3 dose groups (300, 1000, or 3000 ppm for
males; 1000, 2500, or 5000 ppm for females) or 1 untreated
group (0 ppm), resulting in 60 rats per group. The doses were
equivalent to average daily doses of approximately 13, 40, or
130 mg/kg to males and 50, 120, or 230 mg/kg to females.
Mice in the chronic core study (total, 240 mice/sex) were
randomly assigned to 1 of 3 treated groups (625, 1250, or
2500 ppm) or 1 untreated group (0 ppm), resulting in 60 mice
per group. The doses were equivalent to average daily doses
of approximately 75, 150, or 315 mg/kg to males and 80, 150,
or 325 mg/kg to females. Doses for the chronic study were
selected based on the results of 13-week subchronic studies.
Rats and mice were individually housed (male mice)
or group-housed (male rats, up to 3/cage; female rats, up
to 5/cage; and female mice, 5/cage). The animals were
given dosed or undosed irradiated powdered NTP-2000 diet
(Zeigler Bros., Inc.) and city tap water ad libitum and kept
in rooms maintained between 69◦ F and 75◦ F, with a rela-
tive humidity of 35–65% and 10–20 changes of filtered air
per hour. Animals were observed twice daily for mortality
and morbidity, and clinical signs of toxicity were recorded
every 4 weeks beginning with week 4. Animals were sacrificed when moribund or after 2 years of exposure. Also, at
6 months, 10 animals per sex randomly selected from each
dosed and control group were sacrificed as interim-sacrificed
animals. A complete necropsy examination was performed
on all animals. Tissues were fixed in 10% neutral buffered
formalin. All of the slides were stained with hematoxylin
and eosin (H&E). In the present study, only the testis and
epididymis were reevaluated. Grading of GEA of the testis
was designated as normal (0), minimal (1) = <10%, mild
(2) = 10–25%, moderate (3) = 25–50%, or severe (4) >50%,
reflecting the extent of affected tubules.
Statistics
We used the NTP’s procedure for trend tests and pairwise comparisons that are based on the Poly-3 methodology (Bailer and Portier, 1988; Portier and Bailer, 1989) for
detecting significance ( p < 0.05).
RESULTS
The incidences of pathological changes in the testis and
epididymis of mice are shown in Table 1. In mice interimsacrificed at 6 months, no testicular or epididymal lesions
were found, even in the high-dose group. At 2 years, the incidence of GEA of the testis was increased in a dose-related
manner ( p < 0.01), and the incidences in the 2 highest dose
levels (8/50, mid-dose; 14/50, high-dose) were significantly
( p < 0.01) higher than those of the controls (1/50). The numbers of mice with bilateral testicular GEA were comparable
between the control and treated groups, but the number of
mice with unilateral GEA increased in a dose-related manner
(0, 1, 6, and 13 in 0, 625, 1250, and 2500 ppm groups, respectively: p < 0.01). The incidence of SG of the epididymis
increased dose-relatedly (0, 0, 3, and 6 in 0, 625, 1250, and
2500 ppm groups, respectively: p < 0.01), and the incidence
TABLE 1.—Incidences of neoplasms and selected nonneoplastic lesions of the
testis and epididymis in B6C3F1 mice.
Male mice
6-month interim study
Epididymis
Testis
2-year study
Epididymis
Inflammation, chronic active
Sperm granulomac
(Bilateral)
(Unilateral)c
Testis
Germinal epithelium, atrophyc
(Bilateral)
(Unilateral)c
Interstitial cell, adenoma
Sertoli cell tumor, malignant
a
0 ppm
625 ppm
1250 ppm
2500 ppm
(10)a
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(50)
1b
0
0
0
(50)
1
1
0
0
1
(50)
3
0
0
0
(50)
4
3
1
0
0
(50)
7
3d
0
3
(50)
8f
2
6f
1
0
(50)
8
6d,e
1
5e
(50)
14 f
1
13 f
1
0
Number of animals with tissue examined microscopically.
Number of animals with lesions.
Dose-related trend in the incidence was significant ( p < 0.01) by the Poly-3 trend test.
d
These numbers are not consistent with those that appear in NTP TR-516 (National
Toxicology Program, 2004), as the reevaluation in this article was performed after the final
version of the Technical Report was prepared.
e
Significantly different ( p < 0.05) from the vehicle control group by the Poly-3 pairwise
test.
f
Significantly different ( p < 0.01) from the vehicle control group by the Poly-3 pairwise
test.
b
c
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Vol. 33, No. 3, 2005
2-MI-INDUCED SPERM GRANULOMAS IN MICE
315
Figures 2–6
FIGURE 2.—Testis and epididymis of mouse treated with 1250 ppm 2-MI. Germinal epithelial atrophy of the testis and sperm granuloma of the epididymis and
efferent ductule. H&E. Bar = 500 µm. 3.—Magnified view of the testis in Figure 2. Germinal epithelial cells can be seen degenerated or decreased in number and
Sertoli cells occasionally damaged; lumen occupied in places by degenerated spermatozoa. H&E. Bar = 100 µm. 4.—Magnified view of the epididymis in Figure 2.
Sperm granuloma in the caput epididymis (lower right) shows rupture and engorgement of tubules. Note absence of spermatozoa in the tubule downstream of the
lesions. H&E. Bar = 250 µm. 5.—Sperm granuloma in mouse treated with 2500 ppm 2-MI. Ductular epithelium is ruptured, and granulomatous inflammatory reaction
can be seen in the interstitial tissue; mineralization (arrows) is occurring in the granuloma. H&E. Bar = 100 µm. 6.—Sperm granuloma in the caput epididymis
of mouse treated with 2500 ppm 2-MI. Degenerated spermatozoa can be seen in the lumina of dilated tubules. The SG lesion is accompanied by azoospermia
downstream. H&E. Bar = 250 µm.
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316
TABLE 2.—The presence or absence of sperm granuloma (SG) of the epididymis and distribution of germinal epithelial atrophy (GEA) of the testis in
B6C3F1 mice.a
TABLE 4.—Incidences of neoplasms and selected nonneoplastic lesions of the
testis and epididymis in F344/N rats.
Male rats
Distribution of
atrophic tubules
No. of animals
without SG
No. of animals
with SG
12
6
2
7
Near rete or peripheral
Focal/multifocal/diffuse
a
GEA in the testis from all groups (0, 625, 1250, and 2500 ppm).
of the high-dose group was significantly ( p < 0.05) higher
than that of controls. The incidence of unilateral SG also increased in a dose-related manner (0, 0, 3, and 5 in 0, 625,
1250, and 2500 ppm groups, respectively: p < 0.01). Only 1
mouse from the high-dose group that had bilateral SGs also
exhibited bilateral GEA. Eight other mice with unilateral SG
were associated with GEA of the ipsilateral testis. Of 27 mice
that displayed testicular GEA, 18 (67%) mice did not have
SG in either the ipsilateral or contralateral epididymis.
Germinal epithelial atrophy was observed in the testis of
mice (Figure 2). In the atrophic seminiferous tubules, germinal epithelial cells were decreased in number, and Sertoli cells were occasionally affected (Figure 3). Some seminiferous tubules with GEA were associated with luminal
dilatation. The lumen was sometimes occupied by degenerated spermatozoa that replaced the normal epithelial lining (Figure 3). Seminiferous tubules, tubuli recti, and/or rete
testis were sometimes dilated, with cell debris found in the
lumen. If only a few tubules appeared to be associated with
GEA around the rete testis, they were designated tubuli recti
and not counted as GEA. Distribution of atrophic seminiferous tubules varied among animals: focal around the rete
testis, focal away from the rete testis, peripheral, multifocal,
or diffuse. No relationship, however, was detected between
the presence and absence of SG and the distribution of GEA
in the testis (Table 2).
Sperm granulomas were observed in the efferent ductules
and the caput, but not in the corpus and cauda of the epididymis (Figure 2). The wall of ductules of the caput associated with SG was often found to be ruptured, depending on the
sectioning, where spermatozoa were extravasated into surrounding interstitial tissues (Figures 4 and 5). Mononuclear
cell infiltration caused granulomatous inflammatory lesions
(Figures 5 and 6) in which mineralization was occasionally
observed (Figure 5). Epididymal ductules around and/or upstream of the SG were markedly engorged with spermatozoa
(Figures 2, 4, and 5). This intratubular SG was thought to
result in almost complete occlusion, because in lumens of
ductules downstream of the SG, a decrease in the number of
spermatozoa resulted in oligospermia or azoospermia (Figures 2, 4, and 6). Edema around the ductules was sometimes
observed (Figure 6).
TABLE 3.—The grade of germinal epithelial atrophy (GEA) of the testis and
the sperm granuloma (SG) of the epididymis in B6C3F1 mice.a
Grade of GEAb
0–1
2–4
a
n
Incidence of SG
179
21
1 (0.56%)
8 (38%)
Data were taken from all groups (0, 625, 1250, and 2500 ppm).
0, normal; 1, minimal (<10%); 2, mild (10–25%); 3, moderate (25–50%); 4, severe
(>50%).
b
TOXICOLOGIC PATHOLOGY
TANI ET AL.
6-month interim study
Epididymis
Testis
2-year study
Epididymis
Atrophy
Inflammation, chronic
Sperm granuloma
Testis
Atrophyc
Germinal epithelium, atrophyc
Interstitial cell, adenomac
Interstitial cell, hyperplasiac
0 ppm
300 ppm
1000 ppm
3000 ppm
(10)a
(10)
(10)
(10)
(10)
(10)
(10)
(10)
(50)
0b
0
0
(50)
7
2
48
1
(50)
1
1
1
(50)
7
3
45
6
(50)
0
1
1
(50)
15
1
42
5
(50)
1
0
1
(50)
13
1
43
12
a
Number of animals with tissue examined microscopically.
Number of animals with lesions.
Numbers of animals with unilateral and bilateral lesions, with or without multiple lesions, are combined.
b
c
A grading from normal (0) to severe (4) was used to characterize GEA of the testis, depending on the extent of the affected tubules, with no animals diagnosed as severe. Table 3
shows the correlation between the grade of GEA of the testis
and the incidence of SG. Only 1 mouse (0.56%) showed SG
in the epididymis of 179 animals in which there were no
(grade 0) or minimal (grade 1) testicular GEA lesions. On
the other hand, 8 of 21 mice (38%) showing mild (grade 2)
or moderate (grade 3) testicular GEA were found to have
SG in the epididymis. We did not, however, apply a statistical test to this data set, as there were numerous animals
without testicular lesions that would have affected the test
result.
In rats, contrary to mice, SGs occurred very rarely in the
epididymis. No rats exhibited SG in the control group, and
only 1 animal in each treated group was found to have SG
in the epididymis (Table 4). Most rats, regardless of the dose
levels, exhibited interstitial cell hyperplasia or adenoma, or
both, which replaced normal testicular tissues. In rats sacrificed at 6 months, no lesions of the testis and epididymis
occurred.
DISCUSSION
This investigation constitutes the first report showing that
chronic administration of 2-MI can induce SGs in the epididymis of B6C3F1 mice. We, however, could not detect
an increase in the incidence of 2-MI-induced SGs in rat
epididymides.
Mechanisms by which 2-MI induces epididymal SG in
mice are unknown. Indirect evidence in the present study
implied that the most severe presentation of testicular GEA
might occur secondarily to epididymal SG. Twenty of all
27 GEA cases were unilateral, while only 7 mice exhibited
bilateral GEA (Table 1); the incidence of unilateral GEA
increased in a dose-related fashion, whereas that of bilateral
GEA was comparable within the groups. For direct testicular
toxicity, one would generally expect most, if not almost all, of
the lesions to be bilateral. Some chemicals, including methyl
chloride (Working et al., 1985), can induce SGs unilaterally
or bilaterally. In addition, 8 of 9 SG cases were unilateral, with
unilateral, probably ipsilateral, testicular GEA. Only 1 case
had bilateral epididymal SGs, which also exhibited bilateral
testicular GEA. The current information would suggest that
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Vol. 33, No. 3, 2005
2-MI-INDUCED SPERM GRANULOMAS IN MICE
317
TABLE 5.—Chemical-induced sperm granulomas and related lesions reported in rodents.
Drug/operation
Species
Straina
Lesion with testicular degeneration
Benomyl
Rat
SD
Carbendazim
Rat
SD
α-Chlorohydrin
Rat
SU
Methyl chloride
Rat
F344
Lesion without testicular degeneration
L-Cysteine
Rat
SD
Guanethidine
Rat
SD
Lesion with vascular changes
Cadmium chloride Rat
Albino
Lentinan
Rat
SD
Routeb
Gavage
Gavage
Gavage
Inhalation
IP
IP
SC
IV
Dose
∼400 mg/kg
∼800 mg/kg
∼90 mg/kg
3500 ppm × 6 hr/day
∼1000 mg/kg
5 mg/kg
14 mg/kg
∼30 mg/kg
Sitec
Duration
Singled
Singled
Single or 8 days
9 days in total
ED
ED
E (caput)
E (cauda)
∼4 weeks
19 weeks
E (corpus, cauda)
E (cauda)
∼7 days (daily)
5 weeks
E
E
Incidence
16/68e
18/88 f
Unknown
6/6g
Reference
(Hess et al., 1991)
(Nakai et al., 1992)
(Ericsson, 1970)
(Chapin et al., 1984)
6/6h
6/6
(Sawamoto et al., 2003)
(Bhathal et al., 1974)
Unknown
12/12
(Mazzanti et al., 1969)
(Ishii et al., 1980)
a
SD, Sprague–Dawley; SU, Spartan and Upjohn.
IP, intraperitoneally; IV, intravenously; SC, subcutaneously.
c
ED, efferent ductules; E, epididymis.
d
Sacrificed 70 days after gavage.
e
Data taken from treatment groups given 100, 200, and 400 mg/kg of benomyl.
f
Data taken from treatment groups given 100, 200, 400, and 800 mg/kg of carbendazim.
g
At 19 days after commencing exposure to the chemical.
h
Data from 1000 mg/kg group.
b
the less severe GEA can be induced by 2-MI in the absence
of SGs; thus, 2 independent mechanisms for induction of
testicular toxicity may be operating—a direct testicular effect
on the seminiferous epithelium and a more severe atrophy as a
consequence of granuloma induction. The possibility of this
latter mechanism is supported by data for benomyl (Hess
et al., 1991).
Benomyl, a benzimidazole carbamate fungicide, and its
metabolite carbendazim cause occlusion of the efferent ducts
and SGs in the efferent ducts and caput epididymis with secondary long-term seminiferous tubular atrophy in rat testes
(Hess et al., 1991; Nakai et al., 1992; Hess, 1998). Hess and
colleagues have demonstrated that the pathogenesis of the
epididymal lesion is linked to a disturbance in reabsorption
of seminiferous tubule fluid in the efferent ductules and caput epididymis and that part of the tubular atrophy that is
seen occurs due to back pressure of fluid (Nakai et al., 1992;
Hess, 1998). Though whether 2-MI affects reabsorption of
seminiferous tubular fluid in the efferent ductules and caput
epididymis is unknown, 2-MI likely induces SGs primarily
following the leakage of spermatozoa into extraluminal tissue (Stevens and Lowe, 1995) followed by testicular GEA.
Benomyl and carbendazim, also structurally related imidazoles, induce SGs in a similar location in the epididymis
associated with long-term seminiferous tubular atrophy.
A single administration of some kinds of imidazoles, such
as 2-MI, 4-methylimidazole, imidazole (10–300 mg/kg, sc),
or ketoconazole (10–300 mg/kg, gavage), suppresses testicular testosterone secretion, with the weakest effect produced by
2-MI (Adams et al., 1998). Of the imidazoles, ketoconazole
is known to inhibit directly some cytochrome P450 enzymes
responsible for testosterone biosynthesis (Santen et al., 1983;
Sikka et al., 1985); however, no chemicals that decrease blood
testosterone, including ketoconazole, have been reported to
induce SGs directly. A decrease in blood androgen level, thus
is unlikely to be an etiology of SGs.
The formation of SGs by methyl chloride treatment is reported to be blocked by treatment with a chemical with antiinflammatory potential (Chellman et al., 1986), even though
the SGs, unlike those caused by 2-MI, occur in the cauda epididymis (Chapin et al., 1984; Working et al., 1985). Increased
superoxide and decreased nitric oxide are also known to be
involved in SG formation (Chatterjee et al., 2001). That some
inflammatory mediators may participate in 2-MI-induced SG
formation is, therefore, possible.
Other chemicals that have been reported to induce SGs associated with testicular lesions are listed in Table 5. Excessive
doses of L-cysteine induce SGs with a high frequency, as early
as 2 weeks of treatment, in the corpus and cauda epididymis
of the rat, much more frequently than in the caput epididymis
and efferent ductules (Sawamoto et al., 2003). Guanethidine,
an adrenergic neuron blocker, has been reported to induce
SGs in the vas deferens of Sprague–Dawley rats, possibly
by decreasing contractile responses to nerve stimulation that
results in rupture of the vas deferens (Bhathal et al., 1974).
Different parts of the epididymis, however, exert different
functions, including those involving metabolism, hormonereceptor expression (Montiel et al., 2003; Yamashita, 2004),
and anatomical characteristics (Jiang et al., 1994). The presence of a site-specific lesion provides evidence of a probable site-specific mechanism of toxicity. We can, therefore,
assume that mechanisms by which chemicals listed above
cause SGs are likely to be different from mechanism(s) by
which 2-MI induces SGs in the caput epididymis and efferent ductules. Examples exist of chemicals that can induce
epididymal SGs in rats by disturbing blood flow of the epididymis and testis (Table 5). Cadmium chloride was reported
to cause epididymal SGs (Mazzanti et al., 1969) by testicular
epithelial damage induced by an injurious response of the
testicular vasculature (Gunn et al., 1963). Sperm granulomas
induced by lentinan were thought to be caused by the spread
of systemic arteritis to efferent ductules (Ishii et al., 1980).
Vascular damage, however, does not seem to be the cause of
SGs in 2-MI-treated mice, as we did not see any evidence
suggestive of vascular toxicity.
The dose–response relationship for the induction of lesions
provides additional information. In the NTP studies in rats
and mice treated with 2-MI for 15 days and 14 weeks (Chan,
2004), rats given 10,000 ppm (or 560 mg/kg/day as the estimated daily dose level), a 4-fold higher dose than that used
in the present study, showed, at 14 weeks, an elevated incidence of testicular degeneration, and rats given 5000 ppm and
10,000 ppm (300 and 560 mg/kg/day) of 4-methylimidazole
for 14 weeks also displayed elevated incidences of
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318
TANI ET AL.
degeneration in the testis and hypospermia in the epididymis.
Mice, however, treated with up to 10,000 ppm of 2-MI
(1740 mg/kg/day) or 4-methylimidazole (1840 mg/kg/day),
with estimated daily dose levels 3 times higher than those of
rats, did not exhibit any pathological changes in the testis and
epididymis (Chan, 2004). These data suggest that rats may
be more severely affected, if testicular toxicity is induced by
2-MI or 4-methylimidazole via the same mechanism in both
rats and mice. In this study, the maximum dose administered
to rats was 130 mg/kg/day (3000 ppm), while that for mice
was 315 mg/kg/day (2500 ppm), 2.4 times higher than the
rat dosage (National Toxicology Program, 2005). For mice,
perhaps 1740 mg/kg/day was insufficient to induce GEA and
SG in 14 weeks, but 315 mg/kg/day was sufficient to induce
the lesions in 2 years. For rats, 560 mg/kg/day was sufficient
to induce GEA in 14 weeks, but 130 mg/kg was insufficient to
induce the testicular and epididymal lesions in 2 years. This
information might help to explain the reason that only mice
developed SGs in the epididymis in the present study. The
late onset of these changes in mice is unusual. The generally
held wisdom is that male reproductive toxicity in rodents becomes evident within 4 weeks of dosing (Ulbrich and Palmer,
1995). In this study we would assume that for mice a dosing
period of more than 2 weeks should be necessary to induce
SGs, even at a dose level of 10,000 ppm (1740 mg/kg/day),
and that 2 years would be required to induce SGs with a dose
level of 2500 ppm (315 mg/kg/day).
In this study, only 1 male F344 rat in each dosed group
showed epididymal SG. Most rats developed rather spontaneous, common interstitial-cell hyperplasias and/or adenomas (Boorman et al., 1990). Interstitial-cell adenomas
were sometimes extremely large, resulting in compression
of seminiferous tubules that might cause severe GEA. These
interstitial-cell proliferative lesions, therefore, caused difficulty in the assessment of the effect of 2-MI on the testis and
epididymis.
From these data obtained from our study, including putative dose insufficiency, we cannot estimate the effect of 2-MI
on GEA and SG formation. The data do indicate, however,
that 2-MI, if given for 2 years, induces SG in the epididymis of
B6C3F1 mice by unknown mechanisms. Additional studies
are needed to clarify whether inflammatory responses contributed to the formation of sperm granuloma. Although the
literature indicates that rats are generally more prone to develop chemically related sperm granuloma, our experiments,
nonetheless, suggest that mice can develop this side effect
after long-term exposure.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Ms. Jo Anne Johnson
and Drs. June Dunnick and Katsuhiko Yoshizawa from the
NIEHS for their critical review of the manuscript.
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