Potent carcinogenicity of 2,7-dinitrofluorene, an environmental pollutant, for the mammary gland of female SpragueDawley rats
Danuta Malejka-Giganti1,2,3,4,
Gloria A. Niehans1,2,
Mark A. Reichert1,
Kristen K. Bennett1 and
Robin L. Bliss3
1 Veterans Affairs Medical Center, Minneapolis, MN 55417 and
2 Department of Laboratory Medicine and Pathology, University of Minnesota Medical School and
3 University of Minnesota Comprehensive Cancer Center, Minneapolis, MN 55455, USA
 |
Abstract
|
---|
Nitrofluorene compounds are environmental pollutants chiefly from incomplete combustion. This study examined carcinogenicities after one intramammary injection of 2-nitrofluorene (2-NF), 2,7-dinitrofluorene (2,7-diNF) or dimethyl sulfoxide (DMSO) (solvent control) to 30-day-old and of 2-NF, 9-OH-2-NF, 9-oxo-2-NF, 2,7-diNF, 9-oxo-2,7-diNF, 2,5-dinitrofluorene, 9-oxo-2,4,7-trinitrofluorene, N-OH-2-acetylaminofluorene (N-OH-2-AAF) (carcinogen control) or DMSO to 50-day-old female SpragueDawley rats. In 30- and 50-day-old rats 6 and 8 glands/rat, respectively, were injected with 2.04 µmol of compound in 50 µl/gland of DMSO. Whereas all compounds including DMSO yielded combined malignant and benign mammary tumor incidences of 3387% by week 82 after injection, 2,7-diNF produced 100 and 93% incidences significantly (P < 0.001) sooner than did DMSO, i.e. by weeks 2349 and 1848 after treatment of 30- and 50-day-old rats, respectively. Rats treated with 2,7-diNF and 9-oxo-2,7-diNF had significantly (P < 0.0001) and marginally (P = 0.0536) more mammary tumors, respectively, than DMSO-treated rats. In 2,7-diNF-treated rats, the ratio of malignant to benign mammary tumors was 5.4, whereas in all other groups it was <0.5. N-OH-2-AAF, a potent tumorigen when applied to the mammary gland as a solid or in suspension, did not yield the expected tumorigenicity here. The contrasting tumorigenic potencies of 2,7-diNF and N-OH-2-AAF may have been prompted by differences in their solubilities in DMSO. Thus, the poorly soluble 2,7-diNF was slowly absorbed from the injection sites since residues (up to 0.9% of the dose injected) were recovered even after 45 weeks. The data indicate prolonged exposure of the mammary gland to 2,7-diNF and suggest that contamination of the environment with 2,7-diNF, even at low levels, poses substantial carcinogenic risk.
Abbreviations: 2-AAF, 2-acetylaminofluorene; 2-AF, 2-aminofluorene; CIS, carcinomain situ; DMSO, dimethyl sulfoxide; 2,5-diNF, 2,5-dinitrofluorene; 2,7-diNF, 2,7-dinitrofluorene; 2-NF, 2-nitrofluorene; nitro-PAHs, nitrated polycyclic aromatic hydrocarbons; PAHs, polycyclic aromatic hydrocarbons; SD, SpragueDawley; 2,4,7-triNF, 2,4,7-trinitrofluorene.
 |
Introduction
|
---|
Widespread environmental pollution by nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) results from incomplete combustion of fossil fuels and atmospheric nitration and includes their presence in the emissions of diesel and airplane engines, airborne particles and grilled foods (15). The International Agency for Research on Cancer lists several nitro-PAHs, including 1- and 4-nitropyrenes, 1,6- and 1,8-dinitropyrenes, 6-nitrochrysene and 2-nitrofluorene (2-NF) (Figure 1
), as `possibly carcinogenic to humans' (3). Although the hazards from exposure to mutagenic and carcinogenic nitro-PAHs through inhalation, ingestion and skin are recognized (3,610), epidemiological correlations are difficult and not yet established.
Among the more than 200 environmental nitro-PAHs, 1-nitropyrene and 2-NF are prevalent (1,3,68). 2-NF is formed at high levels in diesel, gasoline and kerosene exhausts and is present in river sediments and urban air particles, with especially high levels (3105220 pg/m3) in winter (coal and kerosene heating) (8). In the air of Tokyo, the concentration of 2,7-dinitrofluorene (2,7-diNF) is ~30-fold that of 2-NF and 10-fold that of 1-nitropyrene (11). Other nitrofluorenes found in diesel exhaust include 2,5-dinitrofluorene (2,5-diNF) and 9-oxo derivatives of 2-NF, 2,7-diNF and 2,4,7-trinitrofluorene (2,4,7-triNF). In addition, 9-oxo-2,4,7-triNF is a photoconducting agent, fungicide and an industrial chemical reagent (7). 2-NF and 9-oxo-2-NF are also formed by photo-oxidation of 2-aminofluorene (2-AF) (12,13), which is used in synthetic petroleums and the dye and rubber industries.
Nitroreduction appears to play a key role in the activation of nitrofluorenes to mutagens and carcinogens (7,8,1416). Administration of 2,7-diNF, 2-NF and 2,5-diNF to female SpragueDawley (SD) rats at 1.62 mmol/kg of grain diet for 8 months yielded chiefly mammary gland tumors with incidences of 100, 44 and 12.5%, respectively (17,18). The mammary tumor incidence and multiplicity (~2.5 tumors/rat) induced by 2,7-diNF equaled that by 2-acetylaminofluorene (2-AAF) (18). 2,7-diNF also yielded mammary tumors in male rats (12.5% incidence). After one oral dose of 0.32 mmol of 9-oxo-2,4,7-triNF, a 35% mammary tumor incidence was reported in female SD rats (19). The tumorigenicities of nitrofluorenes for the rat mammary gland described above and that of nitropyrenes (1- and 4-isomers) and dinitropyrenes (1,3- and 1,8-isomers) administered by the s.c., i.p. or oral route (2023) as well as 4-nitropyrene and 6-nitrochrysene by intramammary injection (22,24) suggest the relevance of exposure to nitro-PAHs for breast cancer (10).
The assessment of tumorigenicity by intramammary application is particularly useful since it reveals the capacity of the target tissue to activate a test compound to a tumorigen. Hence, this study was undertaken to examine and compare the tumorigenicities of environmentally significant nitrofluorenes to those of other nitro-PAHs tested by this route previously (2224). The solutions of nitrofluorenes in dimethyl sulfoxide (DMSO) were injected at the dose shown to be tumorigenic for other nitro-PAHs, i.e. at 2.04 µmol/mammary gland. The initial experiment compared the susceptibilities to 2-NF, 2,7-diNF or the solvent DMSO by intramammary injections to 30-day-old rats. Since 50- to 56-day-old rats were also susceptible to mammary tumorigenesis by one intramammary application of polycyclic aromatic hydrocarbons (PAHs) (25,26) or N-OH-2-AAF (27,28), 50-day-old rats were used to compare the susceptibilities to 2-NF, 9-OH-2-NF, 9-oxo-2-NF, 2,7-diNF, 9-oxo-2,7-diNF, 2,5-diNF, 9-oxo-2,4,7-triNF, N-OH-2-AAF (positive control) or DMSO (solvent control).
 |
Materials and methods
|
---|
Chemicals
2-NF, 2,7-diNF and 9-oxo-2-NF were purchased from Aldrich Chemical Co. (Milwaukee, WI), 2,5-diNF and 9-oxo-2,7-diNF from Pfaltz and Bauer (Waterbury, CT) and 9-oxo-2,4,7-triNF from MacKenzie Corp. (Bush, LA). 9-OH-2-NF (29) and N-OH-2-AAF (30) were prepared by published methods. All fluorenyl compounds were recrystallized until >98% pure as determined by HPLC (31). DMSO (Spectranalyzed Grade) was purchased from Fisher Scientific (Pittsburgh, PA). All other chemicals were of the highest quality from commercial sources.
Treatment of rats and tissue removal
Female SD rats (Specific Pathogen Free; Harlan Sprague Dawley, Indianapolis, IN) were housed in a controlled environment with a 12 h light/dark cycle and maintained on Teklad Certified Rodent Diet (Harlan Teklad, Madison, WI) and water ad libitum. An initial group consisted of 20 rats, which on day 30 ± 1 of age were administered 2-NF (six rats), 2,7-diNF (eight rats) or DMSO (six rats). Each of the six left side mammary glands was injected underneath the nipple with 2.04 µmol compound in 50 µl DMSO or with 50 µl DMSO. Thus, the total dose of 2-NF or 2,7-diNF was 12.2 µmol in 300 µl DMSO per rat. The primary group consisted of 135 rats (15 rats/treatment group), which on day 50 ± 1 of age were administered the nitrofluorene compound (2-NF, 9-OH-2-NF, 9-oxo-2-NF, 2,7-diNF, 9-oxo-2,7-diNF, 2,5-diNF or 9-oxo-2,4,7-triNF), N-OH-2-AAF or DMSO. Each of the eight mammary glands (pairs 25) was injected underneath the nipple with 2.04 µmol compound in 50 µl DMSO or with 50 µl DMSO. Thus, the total dose of each compound was 16.3 µmol in 400 µl DMSO per rat. Rats were weighed and palpated for tumors weekly. Rats with palpable tumors or symptoms of distress (e.g. distended abdomen or vaginal bleeding) were decapitated after CO2 inhalation. Mammary glands, tumors or abnormal growths, uterus, liver, spleen and kidney were removed into ice-cold saline, trimmed of extraneous tissue and blotted. Tissue portions were fixed in buffered formalin, sectioned and 56 µm thick sections were stained with hematoxylin and eosin for histopathological examination. Tumors and other tissue abnormalities were classified according to established criteria (32).
Determination of compound residues in the mammary glands
At termination of the rats, yellowish residues of a solid were present in the 2,7-diNF-treated mammary glands. Residues (27 per rat) were removed into 1 ml of water and homogenized with a Polytron homogenizer (type PT 10/35; Kinematica Gmbh, Lucerne, Switzerland) for three bursts of 5 s each at setting 6.0. The homogenate was extracted twice with 1 vol of methyl t-butyl ether. The combined ether extracts were dried over Na2SO4 and evaporated to dryness under a stream of N2. The dried extracts were dissolved in 50100 µl of 2-propanol:methanol (2:1) and aliquots of 10 or 20 µl were analyzed in an HPLC system described previously (31).
Statistical analyses
The effects of treatment on rat body weights were analyzed using the Wilcoxon signed ranks test (33). Cumulative incidences of mammary tumors with respect to time after compound administration were examined using the KaplanMeier method of estimation for survival analysis (34). Data were censored for animals killed with no confirmed mammary tumors. Univariate comparisons between the effects of DMSO (solvent control) and each of the other two (Figure 2
) or eight (Figure 3
) compounds were made using the log rank test; each comparison was adjusted using the the Bonferroni method, with the overall level of significance set to
= 0.05. Total number of mammary tumors present was examined using mean response functions for weighted least squares analysis of variance (35). Contrasts between DMSO- and each of the other eight compound-derived tumors were constructed. P values were adjusted using the Bonferroni method. A univariate analysis of variance was conducted on total weight of mammary tumors yielded by individual compounds (36).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2. Cumulative mammary tumor incidence after intramammary injections of 2-NF, 2,7-diNF or DMSO to 30-day-old rats. Each of the six left side mammary glands was injected underneath the nipple with 2.04 µmol 2,7-diNF (eight rats) or 2-NF (six rats) in 50 µl DMSO or with 50 µl DMSO (six rats). Rats with palpable tumors were killed between 23 and 81 weeks after the injections. Tumor (both malignant and benign) presence was confirmed by microscopic examination. The difference in time for tumor occurrence between 2,7-diNF- and DMSO-treated rats was significant (P < 0.001).
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3. Cumulative mammary tumor incidence after intramammary injections of nitrofluorenes, N-OH-2-AAF or DMSO to 50-day-old rats. Each of the eight mammary glands (pairs 25) was injected underneath the nipple with 2.04 µmol of the nitrofluorene compound listed or N-OH-2-AAF in 50 µl DMSO or with 50 µl DMSO (15 rats/compound). Rats with palpable tumors were killed between 18 and 82 weeks after the injections. Tumor (both malignant and benign) presence was confirmed by microscopic examination. The difference in time for tumor occurrence between 2,7-diNF- and DMSO-treated rats was significant (P < 0.001).
|
|
 |
Results
|
---|
The effective dose for mammary tumor induction by 4-nitropyrene or 6-nitrochrysene by intramammary injections was 2.04 µmol in 100 µl DMSO for each of the six left side mammary glands of 30-day-old rats (22,24). In our experience, however, the above volume was too large to be retained at the injection site and, hence, it was reduced to 50 µl of DMSO containing 2.04 µmol/gland of compound. The tumorigenicities of 2-NF, 2,7-diNF or the solvent DMSO were examined in 30-day-old rats with the above dosage. However, occasional tumors in the contralateral side suggested diffusion of compounds. Therefore, 50-day-old rats with fully developed mammary glands were injected bilaterally into mammary glands 25 with 2.04 µmol/50 µl/gland. The extreme gland pairs 1 and 6 were avoided due to their proximity to other glands. The tumorigenicities of the nitrofluorene compounds 2-NF, 9-OH-2-NF, 9-oxo-2-NF, 2,7-diNF, 9-oxo-2,7-diNF, 2,5-diNF and 9-oxo-2,4,7-triNF were compared with that of N-OH-2-AAF and the solvent control. The solubilities of the test compounds determined in DMSO at room temperature were as follows: N-OH-2-AAF (400 mM) > 9-oxo-2,4,7-triNF (300 mM) > 2-NF or 9-OH-2-NF (100 mM) > 2,5-diNF (50 mM) > 9-oxo-2,7-diNF (33 mM) > 9-oxo-2-NF (25 mM) > 2,7-diNF (11 mM). Hence, to reach the desired concentration of 40.8 mM, the latter three compounds were dissolved in DMSO at 6080°C and injected at ~37°C, which did not affect their stability as determined by HPLC. After injection of the solution containing 2,7-diNF, a precipitate formed within the injection site. At termination of the rats, yellowish particles were found in the mammary glands of both age groups treated with 2,7-diNF. The presence of 2,7-diNF at 0.030.88% of the dose (2.04 µmol) applied per mammary gland was determined between 18 and 45 weeks after injection by HPLC analysis. Thus, the prolonged presence of 2,7-diNF at the injection sites coincided with its poor solubility in DMSO relative to the other fluorene compounds tested and indicated its slow absorption. On the other hand, rapid absorption of N-OH-2-AAF, the most soluble of the compounds tested, had apparently occurred since its intramammary injection produced an early systemic effect, evident as weight loss, in contrast to the weight gains observed in the other groups. Both the weight gains of 6.47 ± 4.42 (median 5, P = 0.0001) and 9.33 ± 6.77 (median 8, P = 0.0002) in 9-oxo-2,4,7-triNF- and DMSO-treated rats, respectively, and the weight loss of 6.33 ± 9.25 (median 3, P = 0.0110) in N-OH-2-AAF-treated rats on day 4 after injection were significant. A week later and during the remaining period of the experiment there were no significant differences in the weight gains among the treatment groups.
The cumulative mammary tumor incidences, i.e. the number of rats with tumors confirmed by microscopic examination with respect to time (weeks) after compound application to mammary glands of 30- (Figure 2
) and 50-day-old (Figure 3
) rats, were evaluated in three separate analyses: combined malignant and benign tumors, malignant tumors only and benign tumors only. In both age groups, 2,7-diNF yielded combined malignant and benign mammary tumors significantly (P < 0.001) sooner (incidence of ~100% by week 48) than did the solvent DMSO (incidence of 67% by week 82). In 50-day-old rats 2,7-diNF also yielded malignant mammary tumors significantly (P < 0.001) sooner than did the solvent. However, there were no significant differences between 2,7-diNF and DMSO in the cumulative incidences of benign mammary tumors or between DMSO and the other fluorene compounds (Figures 2 and 3
) in any of the cumulative tumor incidences. The latent periods of 2349 and 1848 weeks for mammary tumors induced by 2,7-diNF in 30- and 50-day-old rats, respectively, were similar (log rank test statistic 0.959, P = 0.327), whereas the latent periods of tumors in the DMSO groups were significantly longer in the 30- than the 50-day-old rats, i.e. 7681 versus 3182 weeks (log rank test statistic 4.488, P = 0.034). The data indicate a similar susceptibility of both age groups to the carcinogenicity of 2,7-diNF and a lower susceptibility to tumorigenicity of DMSO at the younger age.
After injections of 2-NF, 2,7-diNF or DMSO to the six left mammary glands of 30-day-old rats, tumors were found in both the left and right side mammary glands (Table I
), which suggested diffusion of the injected solution or a systemic effect due to absorption. However, the malignant tumors induced by 2,7-diNF were exclusively at the site of injection. Their marked multiplicity coincided with the largest total tumor weight in the 2,7-diNF-treated rats. The microscopic evaluation of tumors induced by 2,7-diNF showed a relatively large number of mammary adenocarcinomas and the presence of fibrosarcomas and carcinosarcomas (Table II
). One malignant mammary tumor in the 2-NF-treated group was ductal carcinoma in situ (CIS). In addition, one rat in this group had a hepatocellular carcinoma, suggesting a systemic carcinogenic effect of 2-NF after intramammary injection. In the benign tumor category, fibroadenomas were more abundant than adenomas in all three treatment groups. Ductal and/or lobular hyperplasia were present in a greater number of mammary glands of 2-NF- or DMSO- than 2,7-diNF-treated rats. This coincided with an earlier termination of the latter group due to the tumor presence and hence suggested that the mammary gland hyperplasia is age-related. Likewise, hyperplastic and/or metaplastic changes seen in some of the uteri appear to be age-related.
View this table:
[in this window]
[in a new window]
|
Table II. Pathology of tumors and tissue abnormalities after intramammary injections of 2-NF, 2,7-diNF and DMSO to 30-day-old rats
|
|
In the primary experiment, using 50-day-old rats for intramammary injections (15 rats/compound), a trend similar to that observed above regarding the multiplicity of malignant mammary tumors induced by 2,7-diNF was noted (Table III
). Statistical analyses showed that 2,7-diNF yielded significantly (P < 0.0001) more tumors (combined malignant and benign) and 9-oxo-2,7-diNF produced marginally (P = 0.0536) more tumors than DMSO. The multiplicity of tumors in 2,7-diNF- and 9-oxo-2,7-diNF-treated rats was consistent with the large total mammary tumor weights in these two groups. However, statistical analysis of the total mammary tumor weights (available for 74 of 89 rats with confirmed mammary tumors) showed no significant differences among the individual compounds [F(8,65) = 5.34, P = 0.6015]. The small sample sizes prevented separate evaluations of weight differences for malignant and benign tumors. The most abundant malignant tumors induced by 2,7-diNF were mammary adenocarcinomas which, together with ductal CIS and carcinosarcomas (the histopathology of representative tumors is shown in Figure 4
), exceeded by 5.4-fold the total number of benign mammary tumors in this group (Table IV
). In all other groups the numbers of benign mammary tumors were greater than those of malignant tumors, yielding the following order of decrease in the malignant to benign tumor ratios: 9-oxo-2,7-diNF (0.47) > 9-oxo-2-NF (0.44) > 9-OH-2-NF (0.29) > 9-oxo-2,4,7-triNF = N-OH-2-AAF = DMSO (~0.20) > 2,5-diNF = 2-NF (0.14). Ductal and/or lobular hyperplasia was much less abundant in the mammary glands of 2,7-diNF-treated rats, which were killed sooner (1848 weeks) after injection than the remaining groups (2282 weeks). Thus, the hyperplastic changes, which occurred under similar circumstances in the initial experiment (Table II
), appear to be age-related. This abnormality was associated with the presence of numerous cysts of varying sizes filled with a dark yellow, milky liquid. Likewise, the presence of focal or extensive adenomatous hyperplasia and/or cystic squamous metaplasia in the uteri of several rats in each group, except for the 2,7-diNF-treated group wherein only one rat was affected (Table IV
), appears to be age-related. Systemic carcinogenic effects of nitrofluorenes were also observed in the uteri of 2-NF- (a superficial adenocarcinoma), 9-oxo-2-NF- (a leiomyosarcoma), 9-oxo-2,4,7-triNF- (a stromal sarcoma) and N-OH-2-AAF-treated (an invasive and in situ adenocarcinoma) rats. One rat in each group of 2-NF-, 2,7-diNF-, 2,5-diNF- and DMSO-treated rats developed nephroblastoma/sarcoma-type large tumors (ranging in weight from 22 to 86 g) in one or both kidneys.
View this table:
[in this window]
[in a new window]
|
Table III. Mammary tumorigenicities of the nitrofluorenes, N-OH-2-AAF and DMSO after intramammary injections to 50-day-old rats
|
|

View larger version (110K):
[in this window]
[in a new window]
|
Fig. 4. Representative malignant mammary tumors after intramammary injections of 2,7-diNF to 50-day-old rats. (A) Adenocarcinoma. Note the infiltrating fused neoplastic glands (x320). (B) Carcinosarcoma. Note the mixed differentiation, with malignant glands interspersed with malignant spindle cells (x160). (C) Ductal CIS. Note the intraductal proliferation of malignant cells (x80).
|
|
View this table:
[in this window]
[in a new window]
|
Table IV. Pathology of tumors and tissue abnormalities after intramammary injections of the nitrofluorenes, N-OH-2-AAF and DMSO to 50-day-old rats
|
|
 |
Discussion
|
---|
After exposure of 30-day-old rats by intramammary injections to 2-NF, 2,7-diNF or DMSO and of 50-day-old rats to 2-NF, 9-OH-2-NF, 9-oxo-2-NF, 2,7-diNF, 9-oxo-2,7-diNF, 2,5-diNF, 9-oxo-2,4,7-triNF, N-OH-2-AAF or DMSO at 2.04 µmol of compound in 50 µl DMSO per gland, only 2,7-diNF induced mammary tumors (malignant or combined malignant and benign) significantly sooner than DMSO. Thus, the latent periods for mammary tumors in 2,7-diNF-treated rats at the age of 30 or 50 days were 2349 or 1848 weeks, respectively, and those in DMSO-treated rats were 7681 or 3182 weeks, respectively. In addition, a marked multiplicity of malignant mammary tumors was observed at the injection sites of 2,7-diNF in both age groups. In 50-day-old rats, the total number of malignant and benign mammary tumors induced by 2,7-diNF was significantly greater and that induced by 9-oxo-2,7-diNF was marginally greater than that induced by DMSO. The greater numbers of tumors were consistent with the larger total tumor weights in the above groups. The carcinogenicity of 2,7-diNF was also remarkable in that it induced a 5.4-fold greater number of malignant than benign mammary tumors whereas all other treatments yielded greater numbers of benign than malignant tumors. The carcinogenic potency of 2,7-diNF for the mammary gland of 30- and 50-day-old SD rats was very similar to that of 6-nitrochrysene and greater than that of 4-nitropyrene tested at the same dose level per gland of 30-day-old CD rats (an outbred strain equivalent to SD rats) in terms of tumor incidences, multiplicities and spectrum of malignancies (22,24). In these studies, DMSO was also found to be tumorigenic in that within 40 and 77 weeks after intramammary injections at 100 µl per gland, 17 and 21% of rats, respectively, developed mammary tumors. Whereas, these tumors were chiefly fibroadenomas (22,24), occasional adenocarcinomas were also present (22,37). It is thus possible that the tumorigenicity of DMSO masked the activities of 1- and 2-nitropyrene in the earlier study (22) and of nitrofluorenes (except for the potent carcinogen 2,7-diNF) herein. In our study, the latency period for development of mammary tumors with DMSO was significantly longer in 30- than 50-day-old rats. Hence, because of their delayed susceptibility to tumorigenic effects of the solvent and/or to spontaneous mammary tumors, which are recognized in the SD strain (38,39), the use of younger rats appears advantageous for assessment of tumorigenicities of suspected carcinogens.
Since N-OH-2-AAF has been shown to be a potent tumorigen for the rat mammary gland after one intramammary application (27,28), it was used as a positive control herein. In the earlier assays, N-OH-2-AAF was applied to 50-day-old rats as a solid at 6.7 µmol to each of three glands (20 µmol/rat) (27) or in a suspension at 2.5 µmol in 50 µl of trioctanoin:acetone (3:1) to each of six glands (15 µmol/rat) (28). Whereas the dose of 2.04 µmol/gland (16.3 µmol/rat) used herein was similar to that reported above, N-OH-2-AAF injected in DMSO failed to elicit a significantly different tumorigenic response from the solvent itself. N-OH-2-AAF yielded an early systemic effect which was evident as a significant weight loss 4 days after injection and indicated its rapid absorption from the injection sites. However, the absorbed dose was insufficient for a significant systemic carcinogenic effect, which was shown previously after one i.p. injection of a 10-fold larger dose (160 µmol) (40). The data indicate that differences in the solubilities of the test compounds in DMSO affected their tumorigenicities at the site of injection. Thus, the poorly soluble 2,7-diNF, which was still detectable at the sites 45 weeks after injection, and the easily soluble N-OH-2-AAF, which was rapidly absorbed, produced potent and weak responses, respectively, in the treated mammary glands. Therefore, comparisons of carcinogenic potentials among structurally diverse compounds might be better accomplished after intramammary applications of their equimolar amounts in the form of solids or suspensions, as practiced in numerous earlier studies (2528).
Potent mammary carcinogenicity of 2,7-diNF by intramammary injections to 30- and 50-day-old female rats reported herein indicates the capacity of the mammary gland of both age groups for activation of this compound. Although activation of 2,7-diNF to a DNA adduct(s) in the mammary gland has not yet been examined, a recent study from this laboratory showed the capacity of the target tissue to reduce this compound to 2-amino-7-NF in vitro (41). The rate of the reduction of 2,7-diNF was greater than that of 2-NF to 2-AF. Nitroreduction of nitro-PAHs is considered to be a principal route to species yielding critical DNA adducts in tumorigenesis (4244). After administration of 2,7-diNF to male Wistar rats, two major adducts were detected by 32P-post-labeling of the liver DNA (45). Whereas the identities of these adducts have not been established, it was presumed that nitroreduction by intestinal microflora contributed to their formation since high levels of the known 2-AF-derived DNA adduct were determined after oral intubation of 2-NF. 2,7-DiNF was an ~2-fold more potent initiator of liver tumorigenesis than 2-NF (45), which is consistent with its greater ease of nitroreduction shown in our studies (31,41).
In addition to adduction to DNA of the reduced nitro group-derived nitrenium ions of nitro-PAHs (42), nitroreduction via one and/or two electron pathways leads to superoxide anion radicals and H2O2, which, in the presence of superoxide dismutase and iron, yield hydroxyl radicals and other oxidants capable of damaging DNA (46,47). These oxidants may also react with lipids yielding DNA-reactive lipid peroxidation products (48). Generation of these oxidants in the lipid-rich target tissue during the prolonged presence of 2,7-diNF at the sites of intramammary injections shown herein would increase the chances of their interaction with mammary gland DNA.
Likewise, DNA-damaging reactive O2 species might be generated at the target site by polymorphonuclear leukocytes stimulated to undergo a respiratory burst in response to the presence of foreign matter (49).
Investigation of the fate and activation of the potent mammary gland carcinogen 2,7-diNF in its target tissue, including identification of its DNA adducts, is a prerequisite to biomonitoring in exposed populations. Biomonitoring of 2,7-diNF-derived DNA adducts in the population of Tokyo, where the levels of this carcinogen in the air are particularly elevated (11) and the age-adjusted death rates from breast cancer are high relative to non-urban areas of Japan (50), may lead to important epidemiological correlations. Whether 2,7-diNF contributes to the high levels of nitro-PAHs in grilled foods (4,5) remains to be determined.
 |
Acknowledgments
|
---|
This study was supported by Biomedical Research Funds, the US Department of Veterans Affairs and a grant from the National Cancer Institute (CA-28000), the US Public Health Service.
 |
Notes
|
---|
4 To whom correspondence should be addressed at: VA Medical Center, 1 Veterans Drive (151), Minneapolis, MN 55417, USAEmail: malej001{at}maroon.tc.umn.edu 
 |
References
|
---|
-
Schuetzle,D. (1983) Sampling of vehicle emissions for chemical analysis and biological testing. Environ. Health Perspect., 47, 6580.[ISI][Medline]
-
Ramdahl,T., Zielinska,B., Arey,J., Atkinson,R., Winer,A.M. and Pitts,J.N.Jr (1986) Ubiquitous occurrence of 2-nitrofluoranthene and 2-nitropyrene in air. Nature, 321, 425427.[ISI][Medline]
-
IARC (1989) Monographs on the Evaluation of the Carcinogenic Risks to Humans, Vol. 46, Diesel and Gasoline Engine Exhausts and Some Nitroarenes. IARC, Lyon.
-
Kinouchi,T., Tsutsui,H. and Ohnishi,Y. (1986) Detection of 1-nitropyrene in yakitori (grilled chicken). Mutat. Res., 171, 105113.[ISI][Medline]
-
Schlemitz,S. and Pfannhauser,W. (1996) Analysis of nitro-PAHs in food matrices by on-line reduction and high performance liquid chromatography. Food Addit. Contam., 13, 969977.[ISI][Medline]
-
Möller,L., Lax,I. and Eriksson,L.C. (1993) Nitrated polycyclic aromatic hydrocarbons: a risk assessment for the urban citizen. Environ. Health Perspect., 101 (suppl. 3), 309315.
-
Beije,B. and Möller,L. (1988) 2-Nitrofluorene and related compounds: prevalence and biological effects. Mutat. Res., 196, 177209.[ISI][Medline]
-
Möller,L. (1994) In vivo metabolism and genotoxic effects of nitrated polycyclic aromatic hydrocarbons. Environ. Health Perspect., 102 (suppl. 4), 139146.
-
Rosenkranz,H.S. (1996) Mutagenic nitroarenes, diesel emissions, particulate-induced mutations and cancer: an essay on cancer-causation by a moving target. Mutat. Res., 367, 6572.[ISI][Medline]
-
El-Bayoumy,K. (1992) Environmental carcinogens that may be involved in human breast cancer etiology. Chem. Res. Toxicol., 5, 585590.[ISI][Medline]
-
Matsushita,H. and Iida,Y. (1986) Application of capillary gas chromatography to environmental analysis. J. High Resolut. Chromatogr. Chromatogr. Commun., 9, 708711.[ISI]
-
Okinaka,R.T., Nickols,J.W., Whaley,T.W. and Strniste,G.F. (1984) Phototransformation of 2-aminofluorene into N-oxidized mutagens. Carcinogenesis, 5, 17411743.[Abstract]
-
Strniste,G.F., Nickols,J.W., Okinaka,R.T. and Whaley,T.W. (1986) 2-Nitrofluoren-9-one: a unique mutagen formed in the photooxidation of 2-aminofluorene. Carcinogenesis, 7, 499502.[Abstract]
-
McCoy,E.C., Rosenkranz,E.J., Rosenkranz,H.S. and Mermelstein,R. (1981) Nitrated fluorene derivatives are potent frameshift mutagens. Mutat. Res., 90, 1120.[ISI][Medline]
-
Vance,W.A., Wang,Y.Y. and Okamoto,H.S. (1987) Disubstituted amino-, nitroso-, and nitrofluorenes: a physicochemical basis for structureactivity relationships in Salmonella typhimurium. Environ. Mutagen., 9, 123141.[ISI][Medline]
-
Fu,P.P. (1990) Metabolism of nitro-polycyclic aromatic hydrocarbons. Drug Metab. Rev., 22, 209268.[ISI][Medline]
-
Miller,J.A., Sandin,R.B., Miller,E.C. and Rusch,H.P. (1955) The carcinogenicity of compounds related to 2-acetylaminofluorene II. Variations in the bridges and the 2-substituent. Cancer Res., 15, 188199.[ISI]
-
Miller,E.C., Fletcher,T.L., Margreth,A. and Miller,J.A. (1962) The carcinogenicities of derivatives of fluorene and biphenyl: fluoro derivatives as probes for active sites in 2-acetylaminofluorene. Cancer Res., 22, 10021014.[ISI]
-
Huggins,C. and Yang,N.C. (1962) Induction and extinction of mammary cancer. Science, 137, 257262.[ISI][Medline]
-
Hirose,M., Lee,M.-S., Wang,C.Y. and King,C.M. (1984) Induction of rat mammary gland tumors by 1-nitropyrene, a recently recognized environmental mutagen. Cancer Res., 44, 11581162.[Abstract]
-
El-Bayoumy,K., Rivenson,A., Johnson,B., DiBello,J., Little,P. and Hecht,S.S. (1988) Comparative tumorigenicity of 1-nitropyrene, 1-nitrosopyrene, and 1-aminopyrene administered by gavage to Sprague-Dawley rats. Cancer Res., 48, 42564260.[Abstract]
-
Imaida,K., Hirose,M., Tay,L., Lee,M.-S., Wang,C.Y. and King,C.M. (1991) Comparative carcinogenicities of 1-, 2-, and 4-nitropyrene and structurally related compounds in the female CD rat. Cancer Res., 51, 29022907.[Abstract]
-
Imaida,K., Lee,M.-S., Wang,C.Y. and King,C.M. (1991) Carcinogenicity of dinitropyrenes in the weanling female CD rat. Carcinogenesis, 12, 11871191.[Abstract]
-
El-Bayoumy,K., Rivenson,A., Upadhyaya,P., Chae,Y.-H. and Hecht,S.S. (1993) Induction of mammary cancer by 6-nitrochrysene in female CD rats. Cancer Res., 53, 37193722.[Abstract]
-
Sinha,D. and Dao,T.L. (1974) A direct mechanism of mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene. J. Natl Cancer Inst., 53, 841846.[ISI][Medline]
-
Cavalieri,E., Rogan,E., Higginbotham,S., Cremonesi,P. and Salmasi,S. (1988) Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of fluorinated derivatives of benzo[a]pyrene and 3-methylcholanthrene. J. Cancer Res. Clin. Oncol., 114, 1622.[ISI][Medline]
-
Malejka-Giganti,D., Rydell,R.E. and Gutmann,H.R. (1977) Mammary carcinogenesis in the rat by topical application of fluorenylhydroxamic acids and their acetates. Cancer Res., 37, 111117.[Abstract]
-
Allaben,W.T., Weeks,C.E., Weis,C.C., Burger,G.T. and King,C.M. (1982) Rat mammary gland carcinogenesis after local injection of N-hydroxy-N-acyl-2-aminofluorenes: relationship to metabolic activation. Carcinogenesis, 3, 233240.[ISI][Medline]
-
Pan,H.-L. and Fletcher,T.L. (1958) Derivatives of fluorene. V. 9-Hydroxyfluorenes; reduction of fluorenes in the presence of aralkylideneamino groups. J. Org. Chem., 23, 799803.[ISI]
-
Miller,E.C., Miller,J.A. and Hartmann,H.A. (1961) N-Hydroxy-2-acetylaminofluorene: a metabolite of 2-acetylaminofluorene with increased carcinogenic activity in the rat. Cancer Res., 21, 815824.[ISI]
-
Ritter,C.L. and Malejka-Giganti,D. (1998) Nitroreduction of nitrated and C-9 oxidized fluorenes in vitro. Chem. Res. Toxicol., 11, 13611367.[ISI][Medline]
-
Rosen,P.P. and Oberman,H.A. (1993) Tumors of the Mammary Gland. Atlas of Tumor Pathology, 3rd Series, Fascicle 7. Armed Forces Institute of Pathology, Washington, DC.
-
Conover,W.J. (1980) Practical Nonparametric Statistics. John Wiley & Sons, New York, NY.
-
Le,C.T. (1997) Applied Survival Analysis. John Wiley & Sons, New York, NY.
-
Agresti,A. (1990) Catagorical Data Analysis. John Wiley & Sons, New York, NY.
-
Montgomery,D.C. (1991) Design and Analysis of Experiments. John Wiley & Sons, New York, NY.
-
Hecht,S.S., Amin,S., Lin,J.-M., Rivenson,A., Kurtzke,C. and El-Bayoumy,K. (1995) Mammary carcinogenicity in female CD rats of a diol epoxide metabolite of fluoranthene, a commonly occurring environmental pollutant. Carcinogenesis, 16, 14331435.[Abstract]
-
McMartin,D.N., Sahota,P.S., Gunson,D.E., Hsu,H.H. and Spaet,R.H. (1992) Neoplasms and related proliferative lesions in control Sprague-Dawley rats from carcinogenicity studies. Historical data and diagnostic considerations. Toxicol. Pathol., 20, 212225.[ISI][Medline]
-
Attia,M.A. (1996) Neoplastic and non-neoplastic lesions in the mammary gland, endocrine and genital organs in aging male and female Sprague-Dawley rats. Arch. Toxicol., 70, 461473.[ISI][Medline]
-
Malejka-Giganti,D. (1981) Activation of certain N-arylacetamides and N-arylacetohydroxamic acids in relation to mammary gland tumorigenesis in the rat. Natl Cancer Inst. Monogr., 58, 6977.[Medline]
-
Ritter,C.L., Decker,R. and Malejka-Giganti,D. (1999) Reductions of 2,7-dinitrofluorenes by rat mammary gland. Proc. Am. Assoc. Cancer Res., 40, 50.
-
Beland,F.A. and Marques,M.M. (1994) DNA adducts of nitropolycyclic aromatic hydrocarbons. In Hemminki,K., Dipple,A., Shuker,D.E.G., Kadlubar,F.F., Segerbäck,D. and Bartsch,H. (eds) DNA Adducts: Identification and Biological Significance. IARC Scientific Publications no. 125, IARC, Lyon, pp. 229244.
-
Herreno-Saenz,D., Evans,F.E., Beland,F.A. and Fu,P.P. (1995) Identification of two N2-deoxyguanosinyl DNA adducts upon nitroreduction of the environmental mutagen 1-nitropyrene. Chem. Res. Toxicol., 8, 269277.[ISI][Medline]
-
Chae,Y.-H., Ji,B.-Y., Lin,J.-M., Fu,P.P., Cho,B.P. and El-Bayoumy,K. (1999) Nitroreduction of 4-nitropyrene is primarily responsible for DNA adduct formation in the mammary gland of female CD rats. Chem. Res. Toxicol., 12, 180186.[ISI][Medline]
-
Möller,L., Cui,X.-S., Torndal,U.-B. and Eriksson,L.C. (1993) Preneoplastic lesions and DNA adduct formation of the airborne genotoxic agents 2-nitrofluorene and 2,7-dinitrofluorene. Carcinogenesis, 14, 26272632.[Abstract]
-
Floyd,R.A. (1990) The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis, 11, 14471450.[ISI][Medline]
-
Frenkel,K. (1992) Carcinogen-mediated oxidant formation and oxidative DNA damage. Pharmacol. Ther., 53, 127166.[ISI][Medline]
-
Dix,T.A. and Aikens,J. (1993) Mechanisms and biological relevance of lipid peroxidation initiation. Chem. Res. Toxicol., 6, 218.[ISI][Medline]
-
Rosen,G.M., Pou,S., Ramos,C.L. Cohen,M.S. and Britigan,B.E. (1995) Free radicals and phagocytic cells. FASEB J., 9, 200209.[Abstract/Free Full Text]
-
Tominaga,S. and Kuroishi,T. (1995) Epidemiology of breast cancer in Japan. Cancer Lett., 90, 7579.[ISI][Medline]
Received March 23, 1999;
revised June 22, 1999;
accepted June 25, 1999.