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.
Introduction
The imidazole derivative, 2-methylimidazole (2-MI1; C4H6N2; 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 caramel-colored syrups (Nishie et al., 1969). This chemical is water-soluble, 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 dose-related germinal epithelial atrophy (GEA) of the testis was also recorded in the original study.
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 relative 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 interim-sacrificed 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 doserelatedly (0, 0, 3, and 6 in 0, 625, 1250, and 2500 ppm groups, respectively: p < 0.01), and the incidence 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).
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 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 ca-put 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 anti-inflammatory 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
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 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.
Footnotes
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.
1
Abbreviations: GEA, germinal epithelial atrophy; H&E, hematoxylin and eosin; 2-MI, 2-methylimidazole; NTP, National Toxicology Program; SG, sperm granuloma.