Comparison of DNA binding between the carcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene

Mutation Research, 301 (1993) 79-85 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00

79

MUTLET 00750

Comparison of DNA binding between the carcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene David K. La and John R. Froines Department of Environmental Health Sciences and UCLA Center for Occupational and Environmental Health, School of Public Health, University of California, Los Angeles, CA 90024, USA (Received 15 May 1992) (Revision received 15 September 1992) (Accepted 15 September 1992)

Keywords: Carcinogenesis; 2,6-Diaminotoluene; 2,6-Dinitrotoluene; DNA adducts; 32p-Postlabelling

Summary We used 32p-postlabelling to compare DNA binding between the potent hepatocarcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene. The two compounds were compared to determine whether differences in DNA binding could partly explain the differences in their carcinogenicity. Fischer-344 rats were administered 1.2 mmol/kg of a compound by single i.p. injection and examined for DNA adduct formation in the liver. Four adducts were detected following administration of 2,6-dinitrotoluene, with a total adduct yield of 13.5 adducted nucleotides per 10 7 nucleotides. Qualitatively identical adducts were also detected after treatment with the derivative 2-amino-6-nitrotoluene. Adduct yields from 2,6-dinitrotoluene were 30 times greater than from 2-amino-6-nitrotoluene. No adducts were observed following treatment with 2,6-diaminotoluene. 2,6-Dinitrotoluene and 2,6-diaminotoluene were also compared for qualitative differences in hepatotoxicity. 2,6-Dinitrotoluene produced extensive hemorrhagic necrosis in the liver, whereas no evidence of hepatocellular necrosis was detected following administration of the latter. The differences between the two compounds in both DNA binding and cytotoxicity were consistent with the differences in their carcinogenicity.

Many aromatic amines and nitroaromatic hydrocarbons are mutagens and carcinogens. The industrial intermediate 2,6-dinitrotoluene (2,6DNT) is a potent animal carcinogen, inducing

Conespondence: Dr. John R. Froines, UCLA Center for Occupational and Environmental Health, School of Public HeaRth, University of California, Los Angeles, CA 90024, USA. Tel. (310) 206-4702; Fax (310) 206-9903. CAS Nos.: 2,6-Diaminotoluene, 823-40-5; 2,6-dinitrotoluene, 606-20-2; 2-amino-6-nitrotoluene, 603-83-8.

hepatocellular carcinomas in rats (Leonard et al., 1987). The reduced derivative 2,6-diaminotoluene (2,6-DAT), however, does not produce tumors in experimental animals (National Cancer Institute, 1980). Both compounds exhibit genotoxicity, although to different extents. 2,6-DNT is potently genotoxic, inducing unscheduled DNA synthesis (Mirsalis and Butterworth, 1982) and demonstrating both initiation and promotion potential (Leonard et al., 1983; Leonard et al., 1986). In contrast, 2,6-DAT exhibits more limited genotoxicity. For example, 2,6-DAT induces morphological transformations in Syrian golden hamster em-

80 bryo ceils (Greene and Friedman, 1980; Pienta et al., 1977), but does not induce unscheduled DNA synthesis (Burmudez et al., 1979; George and Westmoreland, 1991; Mirsalis et al., 1982). 2,6DAT is weakly positive in the rat bone marrow micronucleus test (George and Westmoreland, 1991) and weakly mutagenic in Salmonella (Cunningham et al., 1989; Furlong et al., 1987; George and Westmoreland, 1991; Pienta et al., 1977). The apparent lack of carcinogenicity of 2,6DAT has been the subject of several studies. Based on structure-activity relationships, 2,6-DAT would be expected to exhibit carcinogenic activity. The structural isomer, 2,4-DAT, is both mutagenic and carcinogenic (National Cancer Institute, 1979; World Health Organization, 1987). Studies of related aromatic amine compounds have demonstrated mutagenicity and carcinogenicity to increase when methyl groups are positioned ortho to amino groups (Hecht et al., 1979). The inability of 2,6-DAT to induce tumorigenesis has been shown not to derive from dispositional factors since the compound is rapidly absorbed and extensively metabolized to mutagenic products (Cunningham et al., 1989). A subsequent study showed 2,6-DAT not to induce cellular proliferation in the liver (Cunningham et al., 1991). Studies have shown cell proliferation to be essential in the initiation of chemical carcinogenesis (Cayama et al., 1978; Columbano et al., 1981; Ying et al., 1981). The inability of 2,6-DAT to induce cell proliferation may be an important factor in its apparent lack of hepatocellular carcinogenicity. The covalent binding of chemical compounds or their reactive metabolites to DNA is generally believed to be a key early event in chemical carcinogenesis (Miller, 1978; Wogan and Gorelick, 1985; Yuspa and Poirier, 1988). The replication of modified DNA may result in permanent genetic changes necessary for initiation. Thus, the occurrence of DNA adducts may indicate carcinogenic risk. 2,6-DNT induces extensive DNA adduct formation, in vivo (Dixit et al., 1986; Kedderis et al., 1984; Rickert et al., 1983). In vivo DNA binding by 2,6-DAT has not been previously examined, but one study has found 2,6-DAT to bind DNA, in vitro (Furlong et al., 1987). In this study, we have used 32p-postlabelling to

compare DNA adduct formation between 2,6DNT and 2,6-DAT, in vivo. Qualitative or quantitative differences in DNA binding between the two related compounds could partly explain the differences in their genotoxicity and carcinogenicity. Since cytotoxicity and cellular proliferation may be important elements of carcinogenesis, we have also compared the two compounds for qualitative differences in their toxicity. Materials and methods

Materials

2,6-DAT, 2,6-DNT, and 2-amino-6-nitrotoluene (2A6NT) were purchased from Aldrich Chemical Co., Inc., Milwaukee, WI. Proteinase K was obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN, and T4 polynucleotide kinase from U.S. Biochemical Corp., Cleveland, OH. All other enzymes were from Sigma Chemical Co., St. Louis, MO. Gamma32p-ATP (160 mCi/ml, 7000 Ci/mmol) was purchased from ICN Biomedicals, Inc., Irvine, CA. C-18 reversed phase TLC plates (Whatman) and PEI-cellulose TLC plates were from Alltech Associates, Inc., Deerfield, IL. All other chemicals were of the highest grade commercially available. Animals

Male Fischer-344 rats, weighing 180 to 200 g, were obtained from Simonsen Laboratories (Gilroy, CA). 2,6-DAT, 2,6-DNT, or 2A6NT was dissolved in 100 /zl dimethylsulfoxide (DMSO) and administered to rats by i:p. injection. Control rats received 100 /zl DMSO. After 18 h, rats were killed by cervical dislocation. Livers were excised and stored at -80°C until the time of analysis. DNA isolation

DNA was isolated from liver, as previously described (Gupta, 1984). The DNA concentration was determined spectrophotometrically (50 p~g/ ml D N A = 1 absorbance unit at 260 nm), and adjusted to 0.5 mg/ml. DNA (2/zg) was digested enzymatically to deoxyribonucleoside 3'-phosphates by incubation with micrococcal nuclease and spleen phosphodiesterase (Talaska et al., 1987). Nonadducted nucleotides were then selec-

81 tively d e p h o s p h o r y l a t e d by incubation with 7.5/zg nuclease P1 ( R e d d y and R a n d e r a t h , 1986).

~lP-Postlabelling and chromatography A d d u c t e d nucleotides were end-labelled in a reaction with 100 /~Ci g a m m a - 32P - A T P and T4 polynucleotide kinase to form deoxyribonucleoside 5'- 32P-labelled 3',5'-bisphosphate derivatives ( R e d d y and R a n d e r a t h , 1986). H i g h e r amounts of 32F'-ATP was used to ensure that the radiolabel would be present in excess concentration. The quantitative determination of adduct levels is not affected by excess of 32p-ATP. T h e labelled nucleotides were purified by reversed phase thin layer c h r o m a t o g r a p h y (TLC) using 0.4 M a m m o nium formate, p H 6.0 ( R a n d e r a t h et al., 1984). D N A adducts were then resolved by two-dimensional anion exchange chromatography ( R a n d e r a t h et al., 1984). This step involved separation on PEI-cellulose T L C plates with the following: (a) a solution of 1.8 M lithium formate and 4.25 M urea, p H 3.5 from b o t t o m to top; (b) a solution of 0.56 lithium chloride, 0.35 M TrisHCI, and 6 M urea, p H 8.0 from left to right; and (c) 1.7 M sodium phosphate, p H 6.0 from left to right onto a 2.5 cm wick.

DNA adduct quantitation D N A adducts were detected by screen-intensified a u t o r a d i o g r a p h y and m e a s u r e d by liquid scintillation counting. Alternatively, adducts were m e a s u r e d using the A M B I S radioanalytic imaging system ( A M B I S Systems, Inc., San Diego, CA). T h e two m e t h o d s of detection differed in their efficiencies, but responses by both m e t h o d s were

linear over the range of values m e a s u r e d in our study. T h e two m e t h o d s p r o d u c e d comparable results when D N A adduct levels were expressed as ratios of a d d u c t e d nucleotides to total nucleotides (or relative adduct labelling, RAL). R A L was calculated as follows ( R e d d y and R a n d e r a t h , 1986): RAL cpm in adducted nueleotides specific activity (cpm/pmol)x amt. DNA (pmol)

Results

We used nuclease P l - e n h a n c e d 32p-postlabelling to c o m p a r e D N A binding between 2,6-DNT and 2,6-DAT, in vivo. D N A adduct formation in the liver was detected only following administration of the former c o m p o u n d . After separation of adducted nucleotides by thin layer c h r o m a t o g r a phy, four distinct adduct spots were detected using a u t o r a d i o g r a p h y (Fig. 1). A d d u c t 1 acc o u n t e d for approximately 60% of the total adducts measured, while the proportions for the three minor adducts ranged from l0 to 15%. A l t h o u g h 2,6-DNT was administered in high concentration, additional adducts could not be detected. T h e total adduct yield following administration of 1.2 m m o l / k g 2,6-DNT was 13.5 adducted nucleotides per 107 nucleotides (Table 1). A closely related c o m p o u n d , 2-amino-6-nitrotoluene (2A6NT), p r o d u c e d D N A adducts which were chromatographically identical to those from 2,6-DNT (Fig. 1), but adduct yields from the

TABLE 1 COMPARISON OF DNA ADDUCT FORMATION FROM STRUCUTRAL ANALOGS OF 2,6-DNT ~ Compound

DNA adduct level (RAL× 107) b

2,6-Dinitrotoluene 2,6-Diaminotoluene 2-Amino-6-nitrotoluene

Adduct 1 8.3 _+0.6 n.d. c 0.16 ± 0.02

Adduct 2 2.0 +0.3 n.d. c 0.10 ± 0.02

Adduct 3 1.9 _+0.4 n.d. c 0.08 ± 0.03

Adduct 4 1.3 _+0.3 n.d. c 0.10 ± 0.02

a DNA was isolated from liver of male Fischer-344 rats following single i.p. administration (1.2 mmol/kg) of test compound. b The adduct yields, expressed as means ± standard deviations for 3 animals, were calculated using data obtained from the AMBIS radioanalytic imaging system. c Not detected within the detection limit of one adducted nucleotide per 1011)total nucleotides.

82 latter compound were approximately 30 times higher (Table 1). The chromatograms for both 2,6-DNT and 2A6NT had high background activity. The background could not be reduced by treatment of samples with apyrase or changes in chromatography conditions. 2,6-DAT did not produce detectable levels of D N A adducts even when administered at a twofold greater concentration (Fig. 1). Some D N A adducts derived from aromatic amines are sensitive to nuclease P1 treatment, resulting in signific a n t loss of detectable adducts (Gupta and Earley, 1988). Adduct formation by 2,6-DAT was also examined using the standard labelling (Reddy and Randerath, 1986) and butanol enrichment (Gupta, 1985) procedures to determine if the absence of adducts might be explained by possible losses incurred through the nuclease P1 enrichment process. No adducts from 2,6-DAT were detected using these methods. We also compared the toxicity for 2,6-DNT and 2,6-DAT. Intraperitoneal administration of

B

Fig. 1. Autoradiograms of TLC plates containing JzP-labelled DNA adducts. Liver DNA was isolated from male Fischer-344 rats treated with (A) DMSO or 1.2 mmol/kg of one of the following compounds: (B) 2,6-DAT; (C) 2,6-DNT; (D) 2A6NT. Adducts from 2,6-DNT were exposed to film for 4 h at - 80°C. Screen-enhanced autoradiography for the other compounds was for 24 h. Spots requiring longer exposure for detection have been circled.

0.3 m m o l / k g 2,6-DNT to rats resulted in 50% lethality within two days. 2,6-DAT and 2A6NT did not produce any animal deaths even at 1.2 m m o t / k g . Histopathologic examination of rat liver found 2,6-DNT to induce extensive hemorrhagic necrosis, indicating chemical toxicity (Fig. 2). There was no observable evidence of necrosis in rat liver from 2,6-DAT or 2A6NT despite administration of higher concentrations (Fig. 2). Discussion

D N A adducts are generally promutagenic, and their occurrence may indicate carcinogenic risk. We compared D N A adduct formation between the potent animal carcinogen 2,6-DNT and the apparently noncarcinogenic derivative 2,6-DAT to determine whether the lack of carcinogenicity of the latter could be explained in part by D N A binding. Of the two compounds, only 2,6-DNT induced D N A binding at detectable levels. An earlier finding of D N A binding by 2,6-DAT, in vitro (Furlong et al., 1987) may be explained by dispositional factors. That is, a higher concentration of the compound may reach ceils in culture because competitive processes such as absorption and distribution are avoided. Hence, a greater degree of binding is likely to occur in vitro. Whether 2,6-DAT induces D N A adduct formation in vivo remains unclear. Several short term assays, including an in vivo micronucleus test, have demonstrated 2,6-DAT to be weakly genotoxic (Cunningham et al., 1989; Furlong et al., 1987; George and Westmoreland, 1991; Greene and Friedman, 1980; Pienta et ai., 1977), but other in vivo studies have not found evidence for its genotoxicity (George and Westmoreland, 1991; Mirsalis et al., 1982). If 2,6-DAT binds D N A in vivo, the level of binding would be below the detection limit of one adducted nucleotide per 10 l0 nucleotides. 2,6-DNT induced the formation of four distinct adducts. Although the four adducts could be detected at lower concentrations, a relatively high concentration of 2,6-DNT (1.2 m m o l / k g ) was administered to increase the likelihood Of detecting additional adducts. The doses administered in our study were greater than those employed in the three previous D N A binding studies, but our

83

Fig. 2. Photomicrographs of liver sections from Fischer-344 rats treated with (A) DMSO; (B) 1.2 mmol/kg 2,6-DAT, (C) 0.3 mm~)l/kg 2,6-DNT: (D) 1.2 mmol/kg 2A6NT. Livers were removed 24 h following compound administration, and fixed in 10% buffered formalin. Cross-section blocks were cut mid-lobe, embedded in paraffin, and sectioned (4 #m). H&E, x 250.

adduct yields were found to be significantly lower (Dixit et al., 1986; Kedderis et al., 1984; Rickert et al., 1983). The earlier studies measured total D N A binding, whereas our study measured levels of :specific adducts. The binding in the previous studies may reflect other interactions, such as with the phosphoribose backbone and proteins associated with DNA, as well as to sites on nucleotides which were not detected by our method. Many nitroaromatic compounds and their amine-substituted analogs share common metabolites and adducts (Beland and Kadlubar, 1990). For" example, 2,4-DAT and 2,4-DNT produce qualitatively identical D N A adducts (La and Froines, in press). In our study, 2,6-DNT and

2A6NT produced similar D N A adduct patterns on chromatograms. Bioactivation of 2,6-DNT is thought to involve both oxidative and reductive pathways to form the proximate D N A binding species, 2-hydroxylamino-6-nitrobenzyl alcohol (Rickert et al., 1984). The hydroxylamine is then postulated to be conjugated with sulfate, which subsequently decomposes to form an electrophilic nitrenium ion (Kedderis et al., 1984). There is no obvious explanation for the higher adduct yields of 2,6-DNT relative to 2A6NT. The former may be preferentially converted to this reactive metabolite. The absence of detectable D N A adducts from 2,6-DAT would suggest that either 2,6-DAT is not metabolized to the reactive bind-

84

ing species, or that the ultimate metabolite is not generated in sufficiently large enough yield to produce detectable adduct levels. 2,6-DNT and 2,6-DAT also differed in their cytotoxieity. 2,6-DNT was very cytotoxic to rat liver, while cytotoxicity was not observed with 2,6-DAT. Our observations were consistent with findings from previous studies which examined the toxicity of these compounds. Leonard et al. (1987) found 2,6-DNT to induce increases in enzyme activities of gamma-glutamyl transferase and alanine aminotransferase in rats. Increases in levels of these serum enzymes are indicative of liver injury (Kaplan et al., 1987). Liver toxicity was also demonstrated by Mirsalis and Butterworth (1982) following administration of 100 m g / k g 2,6-DNT to rats. In contrast, 2,6-DAT does not induce cell proliferation in the liver, which suggests lack of cytotoxicity (Cunningham et al., 1991). Cytotoxicity induces cellular proliferation through the compensatory process following cell degeneration and necrosis (Columbano et al., 1981; Ying et al., 1981). Cell proliferation is necessary in the initiation stage of carcinogenesis presumably to fix D N A lesions as permanent mutations (Cayama et al., 1978; Columbano et al., 1981). Regenerative cell proliferation may also be involved in the promotion stage of carcinogenesis (Columbano, 1990). The relatively high adduct yields and potent cytotoxicity of 2,6-DNT were consistent with its carcinogenic potency. The lack of detectable adduct formation and cytotoxicity from 2,6-DAT may be important factors in its lack of carcinogenic activity. References Beland, F.A., and F.F. Kadlubar (1990) Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons, in C.S. Cooper and P.L. Grover (Eds.), Handbook of Experimental Pharmacology, Vol. 94, Part I. Chemical Carcinogenesis and Mutagenesis, SpringerVerlag, Berlin, pp. 267-325. Bermudez, E., D. Tillery, and B.E. Butterworth (1979) The effect of 2,4-diaminotoluene and isomers of dinitrotoluene on unscheduled DNA synthesis in primary rat hepatocytes, Environ. Mutagen., 1,391-398. Cayama, E., H. Tsuda, D.S.R. Sarma, and E. Farber (1978) Initiation of chemical carcinogenesis requires cell proliferation, Nature, 275, 60-62.

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