Tumorigenicity of four optically active bay-region 3,4-diol 1,2-epoxides and other derivatives of the nitrogen heterocycle dibenz[c,h]acridine on mouse skin and in newborn mice

Richard L. Chang1, Alexander W. Wood2, Subodh Kumar3, Roland E. Lehr4, Naohiro Shirai5, Donald M. Jerina5 and Allan H. Conney1

1 Laboratory for Cancer Research, Rutgers, The State University of New Jersey, College of Pharmacy, 164 Frelinghuysen Road, Piscataway,
NJ 08854-8020,
2 Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110,
3 Division of Environmental Toxicology and Chemistry, Center for Environmental Research, State University of New York, College at Buffalo, Buffalo, NY 14222,
4 Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019 and
5 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The nitrogen heterocycle dibenz[c,h]acridine (DB[c,h]ACR) and the enantiomers of the diastereomeric pair of bay-region 3,4-diol 1,2-epoxides as well as other bay-region epoxides and dihydrodiol derivatives of this hydrocarbon have been evaluated for tumorigenicity on mouse skin and in the newborn mouse. On mouse skin, a single topical application of 50 or 200 nmol of compound was followed 10 days later by twice-weekly applications of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate for 20 weeks. DB[c,h]ACR and the four optically pure, bay-region 3,4-diol-1,2-epoxide isomers all had significant tumor- initiating activity. The isomer with (1R,2S,3S,4R) absolute configuration [(+)-DE-2] was the most active diol epoxide isomer. The (–)-(3R,4R)-dihydrodiol of DB[c,h]ACR, the expected metabolic precursor of the bay-region (+)-DE-2, was 4- to 6-fold more tumorigenic than its corresponding (+)-enantiomer. In tumorigenicity studies in newborn mice, a total dose of 70–175 nmol of DB[c,h]ACR or one of its derivatives was injected i.p. on days 1, 8 and 15 of life, and tumorigenic activity was determined when the mice were 36–39 weeks old. DB[c,h]ACR produced a significant number of pulmonary tumors and also produced hepatic tumors in male mice. Of the four optically active bay-region diol epoxides, only (+)-DE-2 and (+)-DE-1 with (1R,2S,3S,4R) and (1S,2R,3S,4R) absolute configuration, respectively, produced a significant tumor incidence. At an equivalent dose, the (+)-DE-2 isomer produced several-fold more pulmonary tumors and hepatic tumors than the (+)-DE-1 isomer. The (–)-(3R,4R)-dihydrodiol, metabolic precursor of the bay-region (+)-DE-2, was strongly active and induced an equal number of pulmonary and hepatic tumors as did DB[c,h]ACR. The (+)-(3S,4S) dihydrodiol was less active. The bay-region (+)-(1R,2S)-epoxide of 1,2,3,4-tetrahydro DB[c,h]ACR was strongly tumorigenic in newborn mice whereas its (–)-(1S,2R)-enantiomer was inactive. This contrasts with the data on mouse skin where both enantiomers had substantial tumorigenic activity. In summary, the bay-region (+)-(1R,2S,3S,4R)-3,4-diol 1,2-epoxide of DB[c,h]ACR was the most tumorigenic of the four optically active bay-region diol epoxides of DB[c,h]ACR on mouse skin and in the newborn mouse. These results with a nitrogen heterocycle are similar to earlier data indicating high tumorigenic activity for the R,S,S,R bay-region diol epoxides of several carbocyclic polycyclic aromatic hydrocarbons.

Abbreviations: DB[c,h]ACR, dibenz[c,h]acridine; (+)-DE-1, (+)-(1S,2R 3S,4R)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[c,h]acridine and (–)-DE-1 its (–)-(1R,2S,3R,4S)-enantiomer;; (+)-DE-2, (+)-(1R,2S,3S,4R)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[c,h]acridine and (–)-DE-2, its (–)-(1S,2R,3R,4S)-enantiomer (the designations 1 and 2 for the diol epoxides indicate that the benzylic 4-hydroxyl group and the epoxide oxygen are cis or trans, respectively); DMSO, dimethyl sulfoxide; 3,4-H2-DB[c,h]ACR, 3,4-dihydrodibenz[c,h]acridine; dihydrodiols; the trans dihydroxydihydro derivatives of dibenz[c,h]acridine in which the hydroxyl groups are at the 1,2-, 3,4- or 5,6-positions; H4-diols, the trans-dihydroxytetrahydro derivatives of dibenz[c,h]acridine in which the hydroxyl groups are at the 1,2- or 3,4-positions; (±)-H4-1,2-epoxide, (±)-1,2-epoxy-1,2,3,4-tetrahydrodibenz [c,h]acridine; (+)-H4-1,2-epoxide, the (+)-(1R,2S)-enantiomer and (–)-H4-1,2-epoxide, the (–)-(1S,2R)-enantiomer; (±)-H4-3,4-epoxide, (±)-3,4-epoxy-1,2,3,4-tetrahydrodibenz[c,h]acridine; PAH, polycyclic aromatic hydrocarbon.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs) are a widespread class of environmental carcinogens formed during the incomplete combustion of organic material (1). Under appropriate combustion conditions, nitrogen may be incorporated into the aromatic ring system to form aza-PAHs (2,3). Some of these aza-aromatic hydrocarbons are found in significant amounts in cigarette smoke, gasoline (petrol) engine exhaust and effluents from the burning of coal (46). Within the past two decades, substantial evidence has been obtained suggesting that, like the PAHs, their aza-analogues are also metabolically activated to diol epoxides according to the bay-region theory (713). The pathway for metabolic formation of the bay-region diol epoxides of the PAHs and the aza-PAHs involves oxidation of a terminal, angular benzo-ring of the hydrocarbon to form an arene oxide, hydration of the arene oxide to form a trans-dihydrodiol, and subsequent epoxidation of the bay-region double bond of the dihydrodiol. These diol epoxides derived from trans-dihydrodiols exist as a pair of diastereomers in which the benzylic hydroxy group is either cis [in diol epoxide (DE)-1] or trans (DE-2) to the epoxide oxygen. Since each diastereomer can be resolved into a pair of enantiomers, there are four possible DE isomers with different absolute configurations.

Earlier studies in our laboratories indicated that the mutagenic activities at the hprt locus in Chinese hamster V79 cells of a large number of the optically active, bay-region DEs from hydrocarbons such as benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[c]phenanthracene (1417) were highly predictive of their tumorigenic activities in mice (1822). In contrast to these results, their mutagenic activities in Salmonella typhimurium TA 98 or TA 100 were not correlated with their tumorigenic activities (1422). In all these studies, the DE with (R,S) diol (S,R) epoxide absolute configuration always had the highest activity in the V79 cell mutagenesis system and had high activity in two mouse tumor model systems (1422). Furthermore, metabolism studies indicated a high degree of stereoselectivity in the formation of this optically active, bay-region DE isomer when the parent hydrocarbons are metabolized by the cytochrome P450-dependent monooxygenase system and epoxide hydrolase (2331).

In this paper we describe the carcinogenic activity in two mouse tumor models of the four possible optically active bay-region 3,4-diol 1,2-epoxides of dibenz[c,h]acridine (DB[c,h]ACR) as well as several other epoxides and epoxide precursors to these DEs for this aza-PAH (Figure 1Go). The parent hydrocarbon, DB[c,h]ACR, has significant mutagenic activity (after metabolic activation) as well as carcinogenic activity (13). DB[c,h]ACR is identical in shape to dibenz [a,j]anthracene but has nitrogen at N-14 as an integral part of both bay regions in this symmetric hydrocarbon (Figure 1Go). Our goal in the present study was to establish whether the (+)-DE-2 isomer of DB[c,h]ACR with (R,S) diol (S,R) epoxide absolute configuration, like the PAH DEs, also has the highest carcinogenic activity among the four possible optically active bay-region DEs.



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Fig. 1. Structures of the nitrogen heterocycle DB[c,h]ACR and 14 of its derivatives studied in this report. Absolute configurations of the optically active isomers of the diastereomeric bay-region 3,4-diol 1,2-epoxides are indicated. In the DE-1 and -2 series, the benzylic hydroxyl group and the epoxide oxygen are cis and trans, respectively. Although not formed metabolically from polyaromatic compounds, the tetrahydro epoxides and their precursors serve as useful model compounds for the study of the biological activity of the PAHs.

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
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Chemicals
DB[c,h]ACR was synthesized and purified as described previously (32). The enantiomerically pure (+)- and (–)-isomers of the diastereomeric pairs of DB[c,h]ACR 3,4-diol 1,2-epoxides were synthesized as described previously (32) from the (+)- and (–)-enantiomers of DB[c,h]ACR 3,4-dihydrodiol of known absolute configuration. Enantiomerically pure (+)- and (–)-H4-1,2-epoxides of DB[c,h]ACR and their racemate were also synthesized as described (32); all stereoisomers were >99% chemically and enantiomerically pure. Procedures for the synthesis of the racemic trans 1,2-, 3,4- and 5,6-dihydrodiols, and the trans 3,4-tetrahydrodiol and 3,4-H2-DB[c,h]ACR have also been reported (32). DB[c,h]ACR H4-3,4-epoxide was synthesized as described (13). Structures of the compounds studied and their absolute configuration are illustrated in Figure 1Go. Compounds were dissolved in dimethyl sulfoxide (DMSO) or acetone and stored in amber vials at –90°C. All manipulations of the compounds were under subdued light. Spectral grade acetone was obtained from Burdick and Jackson, Inc. (Muskegon, MI). DMSO was distilled from calcium hydride under reduced pressure and stored under an atmosphere of argon in amber bottles.

Tumorigenicity studies on mouse skin
Female CD-1 mice at 6 weeks of age (Charles River Breeding Laboratories, Inc., Kingston, NY) were housed in polycarbonate boxes with corn cob bedding and were fed a commercial diet (Purina Laboratory Chow; Ralston Purina Co., St Louis, MO) and water ad libitum. At 7 weeks of age, the mice were shaved on the dorsal surface with electric clippers. Two days later, 30 mice in each treatment group were given a single topical application of compound (50 or 200 nmol) in 200 µl of acetone. Control mice received solvent alone. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (16 nmol/200 µl of acetone) was applied topically twice weekly to each mouse, beginning 9 days after application of the initiator or solvent. Development of skin tumors was recorded every 2 weeks, and papillomas >2 mm in diameter were included in the cumulative total when they persisted for >=2 weeks.

Tumorigenicity studies in newborn mice
CD-1 pregnant mice (Charles River Breeding Laboratories, Inc.) were housed in plastic cages on corn cob bedding. They delivered their litters 5–8 days after arrival. Within 24 h of birth, 10 pups in each litter were given an i.p. injection of compound (one-seventh of the total dose). Subsequent injections were given on days 8 and 15 of life (2/7 and 4/7 of the total dose, respectively). The mice were administered a total dose of 28–175 nmol of compound. Control mice were given three injections of DMSO (5, 10 and 20 µl). The mice were weaned at 25 days of age and killed at 36–39 weeks of age. At necropsy, the major organs of each animal were examined grossly, tumors were counted and tissues were fixed in 10% phosphate-buffered formalin. A representative number of pulmonary tumors and all hepatic tumors were examined histologically. Pathology of the lung tumors was as described previously (33). Most of the hepatic tumors were type A or neoplastic nodules (34,35).


    Results
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 Introduction
 Materials and methods
 Results
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 References
 
Tumorigenicity of DB[c,h]ACR derivatives on mouse skin
The tumor-initiating activity of DB[c,h]ACR, the (+)- and (–)-enantiomers of DB[c,h]ACR trans 3,4-dihydrodiol, the optically pure (+)- and (–)-enantiomers of DE-1 and DE-2 of DB[c,h]ACR, and several benzo-ring derivatives of DB[c,h]ACR on mouse skin are shown in Table IGo. A single initiating dose of 50 or 200 nmol of DB[c,h]ACR produced an average of 0.50 and 1.83 tumors/mouse and a 33% or 60% tumor incidence, respectively. The (–)-(3R,4R)-dihydrodiol of DB[c,h]ACR had tumor-initiating activity equal to that of DB[c,h]ACR, whereas the (+)-(3S,4S)-dihydrodiol had about one-fifth of the tumorigenic activity of DB[c,h]ACR at the same initiating dose. The 1,2-, and 5,6-dihydrodiols, which are not precursors of bay-region DEs, were not tumorigenic on mouse skin. Saturation of the bay-region 1,2-double bond of the DB[c,h]ACR 3,4-dihydrodiol to give the DB[c,h]ACR H4-3,4-diol, which cannot be metabolized to a bay-region 3,4-diol 1,2-epoxide, resulted in little or no tumorigenicity on mouse skin (Table IGo). A comparison of the tumor-initiating activity of the four optically pure bay-region DEs revealed that the (+)-(1R,2S,3S,4R)-3,4-diol 1,2-epoxide-2 [(+)-DE-2] was the most active isomer. A single dose of 50 or 200 nmol of (+)-DE-2 produced skin tumors in 80% of the mice with an average of 2.97 and 4.03 tumors/mouse, respectively (Table IGo). At the lowest dose tested (50 nmol), the (+)-DE-1, (–)-DE-1 and (–)-DE-2 of DB[c,h]ACR had only 25%, 21% and 18%, respectively, of the initiating activity of (+)-DE-2. Comparison of the tumor-initiating activity of the (+)-H4-(1R,2S)-epoxide and (–)-H4-(1S,2R)-epoxide of DB[c,h]ACR indicated that the two enantiomers have essentially equivalent activities as tumor initiators on mouse skin. At the 50 nmol dose tested, these bay-region tetrahydro epoxides produced skin tumors in 80–83% of the mice, and there were ~3.60 tumors/mouse. 3,4-H2-DB[c,h]ACR, the expected metabolic precursor of the highly tumorigenic bay-region H4-1,2- epoxides, along with the above optically active H4-1,2- epoxides, were the most tumorigenic derivatives tested on mouse skin.


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Table I. Tumor-initiating activity of DB[c,h]ACR and its derivatives on mouse skin
 
Tumorigenicity of DB[c,h]ACR derivatives in newborn mice
The tumorigenicities of DB[c,h]ACR and 12 of its derivatives in newborn mice at a total dose of 175 nmol are shown in Tables II and IIIGoGo. By the end of the study (36–39 weeks), only 6% of the control mice had developed pulmonary tumors with an average of 0.13 pulmonary tumors per mouse and only 3% of the control male mice had developed hepatic tumors, with an average of 0.63 hepatic tumors per mouse (Table IIGo). As was observed in initiation–promotion experiments on mouse skin, DB[c,h]ACR and its (–)-(3R,4R)-dihydrodiol had similar tumorigenic activity in newborn mice. Administration of DB[c,h]ACR produced an average of 3.34 pulmonary tumors/mouse and 1.96 hepatic tumors/male mouse. The (+)-(3S,4S)-dihydrodiol was much less active than DB[c,h]ACR in the lung and only somewhat less active than DB[c,h]ACR in the liver. (±)-H4-3,4-diol, (±)-H4-3,4-epoxide and the racemic 1,2- and 5,6-dihydrodiols of DB[c,h]ACR had very weak or no tumorigenic activity (Table IIGo).


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Table II. Tumorigenicity of DB[c,h]ACR and some of its dihydrodiols and tetrahydroepoxides in newborn mice
 

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Table III. Tumorigenicity of DB[c,h]ACR and the enantiomers of the disastereomeric bay-region DB[c,h]ACR 3,4-diol-1,2-epoxides in newborn mice
 
(+)-(1R,2S,3S,4R)-DE-2 and (+)-(1S,2R,3S,4R)-DE-1 were the only two optically active bay-region DEs of DB[c,h]ACR with substantial tumorigenic activity in newborn mice, and (+)-DE-2 was considerably more active than (+)-DE-1 (Table IIIGo). (+)-DE-2 was 12- to 300-fold and 6- to 500-fold more active than the other three isomers in causing pulmonary tumors and hepatic tumors, respectively (Table IIIGo).

(+)-DE-2 and (±)-H4-1,2-epoxide were highly toxic to newborn mice. A total dose of 175 nmol of these compounds killed many mice within 3 weeks, and a reduced dose of 70 nmol of these compounds killed 70% of the mice by the time the experiment was completed at 36–39 weeks. Therefore, we further reduced the total dose to 28 nmol.

In a separate experiment, we treated newborn mice with a total dose of 28 nmol of (+)-DE-2, (–)-DE-2, (+)-H4-(1R,2S)-epoxide or (–)-H4-(1S,2R)-epoxide. Toxicity was still observed for the (+)-DE-2 and (+)-H4-1,2-epoxide groups (43–53% survival; Table IVGo). Although (+)-DE-2 and (+)-H4-(1R,2S)-epoxide were highly active in causing lung and liver tumors, (–)-DE-2 and (–)-H4-(1S,2R)-epoxide had little or no tumorigenic activity (Table IVGo). Notably, on skin both of the enantiomeric H4-epoxides had high and comparable activity.


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Table IV. Tumorigenicity of low doses of optically active dibenz[c,h]acridine diol epoxides and tetrahydroepoxides
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In earlier studies, we evaluated the carcinogenic activities of several racemic bay-region DE isomers of aza-PAHs such as benz[c]acridine, benz[a]acridine and dibenz[a,h]acridine (79). As is the case for the PAHs, the results of these studies clearly established bay-region DEs as ultimate carcinogens for the aza-PAHs. Evaluation of the tumorigenic activity of the present DB[c,h]ACR derivatives on mouse skin (Table IGo) has shown that the (–)-(3R,4R)-dihydrodiol has tumorigenic activity comparable to that of the parent hydrocarbon whereas the 1,2- and 5,6-dihydrodiols were inactive. Saturation of the bay region 1,2-double bond in the 3,4-dihydrodiol to produce the H4-3,4-diol resulted in a loss of activity. All four optically active 3,4-diol 1,2-epoxides were more potent tumor initiators than the (–)-(3R,4R)-dihydrodiol, with the (+)-DE-2 (1R,2S,3S,4R) isomer being the most active. This same isomer was several times more mutagenic than the other three isomers in Chinese hamster V79 cells (13). DB[c,h]ACR is known to be metabolized to its (–)-(3R,4R)-dihydrodiol by cytochrome P-450 and epoxide hydrolase (36). In addition, the (–)-(3R,4R)-dihydrodiol is further metabolized to 3,4-diol-1,2-epoxides (36,37). The importance of the bay region in these skin studies is dramatically emphasized by the observation that 3,4-H2-DB[c,h]ACR and the corresponding H4-1,2-epoxides were the most active compounds tested on skin. The structurally related compound, 3,4-dihydrobenz[c]acridine is the most potent skin carcinogen known for the aza-PAH, benz[c]acridine as well (7).

In the newborn mouse tumor model, DB[c,h]ACR and its derivatives cause pulmonary tumors in both sexes as well as hepatic tumors but only in the males. As on skin, the 3,4-dihydrodiol was active with the (–)-(3R,4R)-enantiomer more active than the (+)-(3S,4S)-enantiomer and comparable to DB[c,h]ACR (Tables I and IIGoGo). Thus on skin as well as in the newborn mouse, only the (R,R)-enantiomer was highly active, as had previously been observed for the corresponding dihydrodiols of the carbocyclic PAHs (19). The 1,2- and 5,6-dihydrodiols of DB[c,h]ACR were inactive. Saturation of the 1,2-double bond (H4-3,4-diol) again resulted in complete loss of activity. The bay-region H4-1,2-epoxide was quite active whereas its non bay-region counterpart (H4-3,4-epoxide) was inactive. The most active compound tested in this set was 3,4-H2-DB[c,h]ACR. On testing the optically active DEs (Table IIIGo), both (+)-(1R,2S,3S,4R)-DE-2 and (+)-(1S,2R,3S,4R)-DE1 were more active than DB[c,h]ACR, with (+)-DE-2 being much more active than (+)-DE-1. When tested at a lower, less toxic dose (Table IVGo), both (+)-DE-2 and the (+)-H4-(1R,2S)-epoxide were highly active. Notably, their enantiomers with 1S-absolute configuration at the reactive C-1 benzylic epoxide center lacked activity. As with DEs of the PAHs (38), R-absolute configuration at the reactive epoxide center plays an important role in expression of tumorigenic activity at least for the present aza-PAHs.

The present study documents for the first time that absolute configuration plays an important role in the expression of tumorigenic activity for an aza-PAH as has been the case for the PAHs (39). The (–)-(3R,4R)-dihydrodiol and the (+)-(1R,2S,3S,4R)-DE-2 of DB[c,h]ACR are identified as proximate and ultimate carcinogens, respectively. It will be of interest to compare the results of the present study with the tumorigenic activity of the related derivatives of the isosteric PAH dibenz[a,j]anthracene, which lacks the N-14 nitrogen. Comparison of the solvolytic reactivity for bay-region DEs of both hydrocarbons shows that nitrogen at N-14 only modestly decreases (2- to 4-fold) rates of reaction (40). This result stems largely from the fact that a positive charge at C-1 derived from opening of the DEs cannot be delocalized directly by resonance on to nitrogen. In contrast, a 3,4-diol 1,2-epoxide of dibenz[a,j]acridine with nitrogen at N-7 is >20-fold less reactive since the nitrogen is in direct conjugation with C-1. These solvolytic studies provide support for earlier theoretical calculations of the ease of formation of a benzylic carbocation, on opening of an epoxide moiety (4144). Such a difference in chemical reactivity presumably accounts for the 10-fold lower mutagenicity of the bay-region DEs of benz[a]acridine compared with benz[c]acridine (45). In the former case, a positive charge at C-1 can be delocalized to nitrogen at N-7.

In summary, both stereochemistry and reactivity play significant roles in determining the tumorigenicity of the aza-PAHs. At present, nothing is known about the role played by these ring nitrogens on binding of DEs to DNA.


    Notes
 
1 To whom correspondence should be addressed Back


    Acknowledgments
 
The authors thank Florence Florek and Keith Williams for their assistance in preparing the manuscript.This study was supported in part by grant CA49756 from the National Institutes of Health.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 8, 2000; revised May 30, 2000; accepted June 12, 2000.