Distinction of mutagenic carcinogens from a mutagenic noncarcinogen in the big blue transgenic mouse.

The aromatic amines 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT) are structural isomers that have been extensively studied for their mutagenic and carcinogenic characteristics. Both compounds are rapidly absorbed after oral administration and are equally mutagenic in the Ames test; however, 2,4-DAT is a potent hepatocarcinogen, whereas 2,6-DAT does not produce an increased incidence of tumors in rats or mice at similar doses. The Big Blue transgenic B6C3F1 mouse carries multiple copies of the lacl mutational target gene. Our studies were designed to determine whether the Big Blue system could be used to detect differences in the vivo mutagenic activity between the carcinogen-noncarcinogen pair 2,4-DAT and 2,6-DAT and to determine whether the in vivo mutagenesis assay results correspond to the rodent carcinogen bioassay results. Male B6C3F1 transgenic mice were exposed to 2,4-DAT or 2,6-DAT at 0 or 1,000 ppm in the diet for 30 and 90 days or to dimethylnitrosamine as a positive control. Mutant frequencies were nearly identical for all three groups at 30 days, while at 90 days the mutant frequency for the hepatocarcinogen 2,4-DAT (12.1 +/- 1.4 x 10(-5)) was significantly higher (p < 0.01) as compared to both age-matched (spontaneous) controls (5.7 +/- 2.9 x 10(-5)) and the 2,6-DAT-exposed group (5.7 +/- 2.4 x 10(-5)). Results from this study demonstrate that the Big Blue transgenic mutation assay can distinguish differences in vivo between the mutagenic responses of hepatic carcinogens ad a noncarcinogen; is sensitive to mutagens through subchronic dietary exposure; and yields a differential response depending upon the length of time mice are exposed to a mutagen.


Introduction
Transgenic mouse mutagenesis assays mutagenic properties of deleterious agents represent a novel approach for assessing the through the use of a stable genomic intemutagenicity of various compounds. gration of the X shuttle vector (XLIZ), Before the availability of these assays, the which carries a lacI target gene and a lacZ mutagenic properties of chemicals were reporter gene (1,2). After treatment of mice often determined using only short term in with the agent in question, genomic DNA vitro tests. The Big Blue assay represents an is isolated from the target organ(s) and the opportunity to examine the in vivo XLIZ is recovered using in vitro phage This paper was developed from a poster that was presented at the 2nd International Conference on Environmental Mutagens in Human Populations held 20-25 August 1995 in Prague, Czech Republic. packaging extracts. Infection of Escherichia coli SCS-8 cells followed by expression of the lacI and lacZ genes permits the detection of phage-carrying mutated lacI genes.
If the normal function of the lacI repressor is disrupted, the lacZ gene product, ,Bgalactosidase, is expressed resulting in the generation of blue plaques on plates containing the chromogenic substrate X-gal (5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside) (1). Scoring the ratio of these blue mutant plaques to colorless nonmutant plaques allows for the quantitative measure of mutant frequency (MO in the tissue of interest. We examined the mutagenic response of the carcinogen-noncarcinogen pair 2,4and 2,6-diaminotoluene (DAT) (Figure 1) in the liver DNA of Big Blue mice after 30 and 90 days of exposure. Both compounds are used extensively in the synthesis of toluene diisocyanates for the production of polyurethane foams and elastomers, and the annual production of both isomers exceeds 100 million pounds (3,4). The compounds are equally mutagenic in the Ames/Salmonella assay in the presence of S9 (5), and both are readily absorbed, metabolized, and excreted (6). However, while the 2,4-DAT isomer was found to be a potent rodent hepatocarcinogen when administered at 100 and 200 ppm in the National Toxicology Program (NTP) bioassay (3), the 2,6-DAT isomer was not carcinogenic when administered at doses up to 100 ppm (4). The Big Blue system has not been evaluated using a wide variety of exposure routes. Most Big Blue studies reported to date have used short-term, high-dose gavage or intraperitoneal exposures (ip) with potent mutagens (7,8). The Big Blue assay has been shown to be sensitive to butadiene administered by inhalation for four weeks (9), and there is one report in which the Big Blue assay was used in a subchronic feeding study of the potent carcinogen, 2-acetylaminofluorene (2-AAF) at 600 ppm (10). Neither of the two DAT isomers examined in our study are extremely potent in vitro mutagens; therefore, a longer-term continuous feeding study was chosen both to increase the sensitivity of the assay and to more closely mimic the dosing regimens used in the standard NTP bioassay. We examined the lacI mutant frequencies induced after exposing male B6C3F1 mice to 2,4-DAT and 2,6-DAT in feed.

Animals and Treatment
Age-matched male, 6-week-old B6C3F, transgenic mice bearing multiple copies of the lacI gene stably inserted into their genome (Big Blue mice) were obtained from Stratagene (Taconic Farms, Germantown, NY). The mice were randomly housed five per cage and were quarantined for 7 days before treatment was initiated. All animals were housed in rooms maintained with 12 hour on-off light cycles. For untreated control and for DAT-exposed animals, powdered NIH-07 feed containing either 0 ppm or the respective DAT at 1,000 ppm was provided ad libitum for 30 or 90 days. Mice were sacrificed at 31 or 91 days. Five animals were assigned to each treatment group. The positive control (DMN-treated) animals received five daily ip injections of 6 mg/kg DMN in saline and were sacrificed 15 days following the last injection. All animals were sacrificed by CO2 sphyxiation and cervical dislocation. Livers were removed, immediately frozen in liquid nitrogen, and stored at -80°C until analysis, as described earlier (11 After centrifugation at 1,000 xg for 10 min, the lower organic phase and protein at the interface were removed and discarded using glass pipettes. This procedure was repeated three times. After the third centrifugation, the upper aqueous phase containing the liver DNA was removed using wide bore pipettes. The DNA was then precipitated by the slow addition of two volumes of ethanol. The DNA was recovered by spooling onto a glass rod and was resuspended in 200 to 500 pl TE buffer. The concentration of DNA was measured spectrophotometrically and adjusted to 0.5 mg/ml with TE buffer (10 mM Tris-HCI and 1 mM EDTA, pH 7.5, autoclaved).

Packaging Genomic DNA
Excision and packaging of the XLIZ vector from genomic DNA was performed using lambda phage packaging extract (Transpack, Stratagene) according to the Stratagene Big Blue instruction manual (12). Rescue efficiency was estimated by plating serial dilutions of the packaging reactions 1 to 2 days before plating to quantitate mutant frequency. All diluted bacteriophages were kept at 40C and were plated within 3 days of packaging.

Plating Assay
The plating procedures generally followed the Big Blue instruction manual (12) Blue plaques, indicating the mutant (lacI+) phenotype, were scored visually using a light box and a red-cellophane transparency to enhance the color contrast of the mutant plaques. All mutant plaques were scored by two individuals. Mutant plaques were cored into individual tubes containing 0.5 ml SM buffer (5.8 g NaCl, 2.0 g MgSO4 [7 H20], 50 ml 1.0 M Tris-HCl, 5 ml 2% gelatin [w/v], made up to 1 liter and pH 7.5 prior to autoclaving) and stored at 4°C for future verification. All mutant plaques were verified by replating isolated phage on 100 mm2 plates in the presence of X-gal.

Effect ofDosing Time
Two of the five positive control animals died during treatment,. apparently due to Environmental Health Perspectives * Vol 104, Supplement 3 * May 1996 DISTINCTION OF MUTAGENIC CARCINOGENS AND NONCARCINOGENS the toxic effects of DMN. All of the DATexposed mice survived the length of the study. The observed Mf in the livers of DMN-treated positive control mice was 31 x 10-5, which is similar to published values using the same treatment regimen (8). Mutant frequencies for the untreated control mice at 10.0 and 5.65 x 10-5 at 30 and 90 days, respectively, were similar to those previously reported (13)(14)(15). The increased mutant frequency observed in 2,4-DAT-treated animals was statistically significantly different (p < 0.01) from agematched controls after feeding the chemical for 90 days, but not following 30 days of chemical exposure (Figure 2A and B). These data indicate a longer dosing regimen may be necessary to observe induced mutations in vivo following treatment with less potent carcinogens. This may be due to the cumulative effects of chemical exposure over a period of 90 days compared to 30 days, to a requirement of a longer time period necessary for the expression of the mutant phenotype in vivo, or a combination of these and other factors.

Effects of Chemical Treatment
The Mf (meanx 10-5± SD; n = 5) at 30 days for control, 2,4-DAT, and 2,6 DAT groups were 10.0 ± 1.9, 9.3 ± 1.4, and 8.7 ± 2.8, respectively (Figure 2A). The Mf at 90 days were 5.7± 2.9, 12.1 ± 1.4, and 5.6 ± 2.4 for control, 2,4-DAT and 2,6-DAT animals, respectively ( Figure 2B). Using a one-way analysis of variance with a least significant difference test, there was a significant difference (p <0.01) in the observed Mf at 90 days between 2,4-DAT treated animals as compared to either the control or the 2,6-DAT-treated animals. The observed increase in Mf at 90 days in the 2,4-DAT treatment group was 2.1-fold over the negative control group Mf at 90 days. As expected, the Mf calculated for the positive control DMN-treated animals was also found to be statistically significantly higher (3.12-fold over control) than all other groups at both time points.

Discussion
The mutagenic isomers 2,4-DAT and 2,6-DAT are of special interest because of their differing carcinogenic response in bioassays conducted by the NTP, even though they exhibit similar mutagenic potencies in Salmonella typhimurium (5,6). The  rats or mice at similar doses. Thus, it was of interest to determine the in vivo mutagenic potential of these isomers in the Big Blue assay system. In order to parallel the NTP 2-year bioassays of 2,4-DAT and 2,6-DAT, we chose to expose the animals to these chemical via the diet. Most Big Blue studies to date have used short-term exposure periods, high-potency mutagens, and parenteral dosing regimens. We reasoned that a continuous exposure study would also be more relevant for comparison to NTP bioassay results. The dose chosen (1,000 ppm) corresponds to the highest nontoxic dose used in a 90-day subchronic study (3,4). The results of the present study demonstrate that 90 days was an appropriate minimum time period for detecting mutations induced by 2,4-DAT administered by feeding. Other investigators have used the dietary route of exposure in the Big Blue model successfully but with the much more potent mutagen, 2-AAF (10). They demonstrated a 3-fold increase in Mf over background after a 28-day subchronic exposure to 600 ppm 2-AAF. The carcinogen, 2,4-DAT, at 1,000 ppm increased the observed Mf in the liver approximately 2-fold over the Mf observed in the agematched control group after 90 days but not after 30 days. The Mf in liver of the 2,6-DAT treatment group also delivered in the diet at 1,000 ppm was not significantly, different from the age-matched control group after either 30 or 90 days of chemical exposure. In this study, the positive control mutagen DMN induced a 3-fold increase in the observed Mf in the liver as compared to the Mf in the age-matched control group (Figure 2). It would appear from these data that longer exposures to some mutagens may be required to produce a detectable mutagenic response in vivo.
This observation should be considered in the design of studies assessing the in vivo mutagenic activity of chemicals of weak or unknown mutagenicity, as well as in the critical evaluation of studies that report negative results following short-term chemical treatment.
In the present study, mutation frequencies were assessed after 30-and 90-day exposures to 2,4-DAT or 2,6-DAT. We demonstrate a 2-fold increase in Mf at the 90-day time point in the 2,4-DAT-treated animals that was significant (p < 0.01) as compared to either the untreated controls or the 2,6-DAT-treated animals. Both these chemicals are in vitro mutagens in the Ames/Salmonella assay, although only 2,4-DAT induces unscheduled DNA synthesis (16). That 2,4-DAT but not 2,6-DAT was found to be mutagenic in vivo is interesting in light of previous studies conducted in this laboratory in which we demonstrated that 2,4-DAT but not 2,6-DAT induces hepatocellular proliferation (17). We postulate that a cytoproliferative effect in target organs could produce an elevated Mf in Big Blue mice as a result of clonal expansion of chemically mutated cells. Cells carrying DNA damage from either 2,4-DAT or 2,6-DAT treatment would yield mutations if forced to replicate; however, hepatocellular proliferation is only induced by 2,4-DAT. The cytotoxicity and compensatory cell proliferation induced by 2,4-DAT treatment leads to mutation fixation during cell proliferation. Such damage effectively increases the mutation rate (mutants/cell/ generation) leading to the elevated mutant frequency (mutants/viable phage) observed in 2,4-DAT-exposed mice at 90 days ( Figure 2B). Such a mechanism could also be involved in the promotion and progression stages of carcinogenesis (18). After 90 days of dietary exposure to 2,4-DAT, the Mf was significantly elevated compared to control ( Figure 2B). This may reflect a sustained proliferative stimulus that may clonally expand mutant cell populations. After a 30-day exposure period, the Mf in 2,4-DAT-treated animals was not elevated, suggesting that longer exposure/expression periods may be necessary to observe the in vivo mutagenic effects of chemicals, the effects of chemicals administered via dietary exposure, or both. The noncarcinogenic isomer 2,6-DAT did not increase the Mf in either the 30-or 90-day animals at a treatment dose identical to the 2,4-DAT treatment dose. Thus, 2,6-DAT may not cause an increase in DNA damage in mouse liver and may only show a mutagenic response in vitro. The proliferative effect has not been observed in 2,6-DAT-exposed animals (17). It is conceivable that DNA damage may have been induced in the liver by exposure to both 2,4-DAT and 2,6-DAT. In the absence of cellular replication in the case of 2,6-DAT-treated mice, such damage may have been repaired with a corresponding reduction of the Mf to background levels. The only known difference between these two in vitro mutagens to explain their widely different mutagenic activity in vivo lies in the abiity of 2,4-DAT to induce hepatocellular proliferation. Chemically induced cell proliferation may therefore be a critical factor in fixation of mutations in vivo. Consistent with a view invoking differential DNA repair, it has been shown recently that enhanced repair of DNA adducts can prevent the carcinogenic effects of methylnitrosourea (19).
In conclusion, this study demonstrates that the Big Blue assay is able to discriminate between the in vivo mutagenic response of two compounds with differing carcinogenic properties but with similar mutagenic activity in S. typhimurium. The major difference between these two chemicals in vivo is an induced cell proliferative response following exposure to 2,4-DAT that does not occur after treatment with 2,6-DAT, suggesting an important role for chemically induced cell proliferation on the mutagenic effect of chemicals. This study also illustrates that the dietary administration of chemicals is an appropriate route of administration for in vivo mutagenesis studies, allowing for longer duration exposures. We believe that these data help to validate this transgenic mouse model as a potential indicator of carcinogenic response and suggest that the Big Blue assay is also useful for mechanistic studies of carcinogenicity.