Chem. Res. Toxicol. 1990, 3, 441-444
441
Identification and Quantitation of 7-(Benzo[ a lpyren-6-yl)guanine in the Urine and Feces of Rats Treated with Benzo[ a Ipyrene Eleanor G. Rogan,**tN. V. S. RamaKrishna,t Sheila Higginbotham,t Ercole L. Cavalieri,? Hyuk Jeong,t Ryszard Jankowiak,* and Gerald J. Small$ Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 5001 1 Received April 20, 1990
The major identified benzo[a] pyrene (BP)-DNA adduct formed by cytochrome P-450 contains
BP bound a t the C-6position to the N-7 position of guanine (BP-N7Gua). This adduct is rapidly depurinated from DNA. When rats were treated with [14C]BP,about 0.02% of the administered dose of B P was excreted as BP-N7Gua in feces and urine within 5 days. Chloroform extracts of the urine and feces were analyzed by high-pressure liquid chromatography. The structure of the adduct was established by cochromatography with electrochemically prepared BP-N7Gua and by fluorescence line narrowing spectrometry. This study represents the first demonstration that BP-N7Gua is formed in vivo in animals treated with BP.
Introduction The carcinogenic activity of chemicals arises from their covalent binding to cellular macromolecules, and modification of DNA presumably plays a central role in tumor initiation. Polycyclic aromatic hydrocarbons (PAH)' require metabolic activation to reactive intermediates that bind to DNA, and cytochrome P-450 plays a major role in this activation. The current view for PAH is that metabolic activation occurs by two main pathways: monooxygenation to yield bay region diol epoxides ( 1 , 2 )and one-electron oxidation to produce radical cations ( 3 , 4 ) . For benzo[a]pyrene (BP) these pathways of activation yield four main adducts, as illustrated in Figure 1. The major identified BP diol epoxide adduct arises from a covalent bond between C-10 of BP diol epoxide and the 2-amino group of dG (BPDEN2dG) ( 2 ) . This adduct is extremely stable in DNA. Another contains BP diol epoxide bound at C-10 to the N-7 of guanine (BPDE-N7Gua) (5-7). This adduct is rapidly depurinated from DNA, leaving an apurinic site. It has been identified by its depurination from DNA treated with BP diol epoxide, but its structure has never been definitively proven. When BP is activated by one-electron oxidation to its radical cation, BP binds at C-6 to the C-8 of dG, forming BP-CMG. This adduct, which is relatively stable in DNA, has been identified in vitro ( 8 ) . BP radical cation also forms an adduct with BP bound at C-6 to the N-7 of guanine (BP-N7Gua). This adduct is rapidly lost from DNA by depurination. It represents at least 75% of the BP adducts formed in the binding of BP to DNA mediated by horseradish peroxidase (8). It is also predominant (1-9 times more abundant than the total amount of stable BP adducts in the DNA) in the cytochrome P-450 catalyzed binding of BP to DNA with rat liver microsomes or nuclei (9). Modification of the N-7 position of guanine in DNA with a number of carcinogens results in products that de-
* To whom correspondence should be addressed. 'University of Nebraska Medical Center. and Iowa State University.
* Ames Laboratory-USDOE
I
,
,
purinate spontaneously, leaving an apurinic site. The released products have been detected in urine. 7Methylguanine was detected in the urine of rats treated with dimethylnitrosamine ( l o ) , and 2,3-dihydro(7guanyl)-3-hydroxyaflatoxinB1 was detected in the urine of rats treated with aflatoxin B, (11). Autrup and Seremet reported the presence of the putative BPDE-N7Gua adduct in the urine of rats treated with BP (7). This is presumably the same adduct detected by Tierney et al. in mice and rats (12). Because the BP-N7Gua adduct is very abundant in the binding of BP to DNA catalyzed in vitro by horseradish peroxidase and cytochrome P-450 (8,9),we decided to look for BP-N7Gua in rats treated with BP by intraperitoneal injection. In this paper we report that BP-N7Gua is excreted in rat feces and urine after treatment of the animal with BP. The structure of the adduct is established by cochromatography with the synthesized BP-N7Gua adduct, UV absorbance spectrum, and fluorescence line narrowing spectrometry (FLNS). FLNS has been successfully applied to a wide variety of biomolecules and to the study of cellular macromolecular damage and chemical carcinogenesis (13). Different stereoisomers of a carcinogenic metabolite which bind to DNA bases can be directly distinguished by laser interrogation of intact DNA (14). Recently, the structure of the major human globin adduct from BP was also identified by FLNS (15).
Experimental Procedures Chemicals. [14C]BP(52 mCi/mmol) was purchased from Amersham (Arlington Heights, IL). Authentic BP-N7Gua was synthesized by anodic oxidation of BP in the presence of 2'deoxyguanosine (8).
Abbreviations: BP, benzo[a]pyrene;BP-N7Gua, 7-(benzo[a]pyren6-y1)guanine;BP-C8dG,8-(benzo[a] yren-6-y1)deoxyguanosine; BPDENZdG, (f)-10~-(deoxyguanosin-hRyl)-78,8a,9a-trihydroxy-7,8,9,~~tetrahydrobenzo[a]pyene;BPDE-N7Gua,(~)-1Ogguanin-7-~1-78,8aeatrihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; dG, deoxyguanosine; FLNS, fluorescenceline narrowing spectrometry;HPLC, high-pressure liquid chromatography; PAH, polycyclic aromatic hydrocarbon(8).
0 1990 American Chemical Society
442
Chem. Res. Toxicol., Vol. 3, No. 5, 1990
Rogan et al. BINDIN6 OF UP TU DNA
RP~ -. ONE-ELECTROM ONE-ELECTROM O X I D A T IION ON OX
N O O X Y 6 E N A T I C 1 1 110NOOXY6ENATIO11
/\ /
0
+
0
PEROXIDASE PEROXIDASE CYTOCHROME
P-450 CYTOCHROME P-450\ + E P O X I D E HYDRASE
P-450
00
no ~
OH
By+ DNA
Figure 1. Formation of BP-DNA adducts by monooxygenation and one-electron oxidation. The terms stable and labile refer to adducts that remain in DNA or are lost by depurination, respectively. Treatment of Rats and Collection of Urine a n d Feces. Eight-week-old male Wistar rats (Eppley Colony) were treated with [“CIBP by intraperitoneal injection. Rats were maintained on a diet of standard laboratory chow and water. In the first experiment the rat was injected with a total of 3.4 pmol of [lrC]BP (91 pCi) in 1.2 mL of trioctanoin split into two equal injections, one on day 0 and a second on day 5. The rat was housed in a metabolism cage supplied only with water to prevent food from contaminating the urine receptacle. The rat was removed to another cage for 2 h each day and allowed to eat. The urine and feces were collected in receptacles cooled in a Dewar flask containing acetone and dry ice. The amount of urine and feces excreted daily was measured, and “C was counted. In the second experiment to determine the time course of BP-N7Gua excretion, the rat was injected with a total of 3.4 pmol of [14C]BP (39 pCi) in 1.2 mL of trioctanoin in one injection, and the urine and feces were collected daily for 5 days. The ‘4c content was counted, and the daily samples were processed separately. Preparation of Samples. In the first experiment, the total urine from 9 days (78 mL) was diluted to 100 mL with H 2 0 and extracted eight times with CHC13 (40-mL aliquots). The protein precipitated from the extractions was collected on a filter and washed with 125 mL of CH30H. The combined organic extracts, which contained approximately 10% of the l‘C in the urine, were evaporated under vacuum, and the residue was analyzed by high-pressure liquid chromatography (HPLC). In the second experiment, the urine collected each day was diluted and treated as described above, except that the volumes were smaller. The fecal samples collected daily were brought to 10 g each with water. The samples were stirred to homogeneous mixtures and then dried by lyophilizationfor 45 h. The dried fecal sample for each day was then extracted with CHC13 (200 mL) in a Soxhlet apparatus for 48 h, and fresh CHCl:, was added after 24 h. The 14Ccontent of each 24-h period was counted; in the second 24 h, the counts were
