- Email: [email protected]
The effects of benzoflavones on polycyclic hydrocarbon metabolism and skin tumor initiation
297
Chem.-Biol. Interactions, 17 (1977) 297--312 © Elsevier/North-Holland Scientific Publishers, Ltd.
THE EFFECTS OF BENZOFLAVONES ON POLYCYCLIC HYDROCARBON METABOLISM AND SKIN TUMOR INITIATION
*
THOMAS J. SLAGA, SARA THOMPSON **, DAVID L. BERRY, JOHN DIGIOVANNI, MONT R. JUCHAU and AURORA VIAJE
Cancer and Toxicology Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 3 7830 and Department of Pharmacology, University of Washington, School of Medicine, Seattle, Wash. 98195 (U.S.A.) (Received October 28th, 1976) (Revision received February 4th, 1977) (Accepted February 28th, 1977)
SUMMARY
The effects of benzoflavones on skin tumor initiation by polycyclic hydrocarbons and epidermal aryl hydrocarbon hydroxylase were investigated. 7,8-Benzoflavone (7,8-BF) was found to be a potent inhibitor of the inhibition of skin tumors by 3-methylcholanthrene (MC) as well as 7,12
298 to be partially related to its ability to inhibit the formation of electrophilic intermediates.
INTRODUCTION
The microsomal enzyme complex of mixed function oxidases is responsible for the detoxification of a variety of compounds, including polycyclic aromatic hydrocarbons [1--6]. Gelboin et al. [7--9] have also proposed that the specific enzyme complex AHH might be responsible for the activation of polycyclic hydrocarbons to toxic and carcinogenic metabolites. This enzyme complex has been found in most mammalian tissues including mouse skin in which it is highly inducible [8,10--15]. Wattenberg and Leong [16,17] reported that i.p. injection of various benzoflavones inhibited polycyclic hydrocarbon induced tumorigenesis in the lung, mammary gland and intestine of rats and mice. 7,8-BF given topically has been shown to inhibit skin t u m o r initiation b y DMBA b u t to enhance t u m o r initiation by BP [8,18--20]. This flavone inhibited the binding of DMBA to DNA, R N A and protein and, to a lesser degree, BP binding to macromolecules [19]. Bowden et al. [20] reported that 7,8-BF inhibited DMBA skin t u m o r initiation, decreased DMBA binding to epidermal macromolecules and reversed the DMBA inhibition of DNA synthesis. They also reported that 7,8-BF stimulated tumorigenesis with DBA and had no effect on the covalent binding of DBA to macromolecules. 5,6-BF slightly inhibited b o t h t u m o r formation and hydrocarbon binding in the case of either DMBA or DBA [20]. The above data led Bowden et al. [20] to hypothesize that methylated polycyclic hydrocarbons are metabolized in a different manner from non-methylated ones b y the epidermal A H H system, especially under the influence of 7,8-BF. In an effort to better understand this relationship, the effects of 7,8-BF and 5,6-BF on epidermal A H H were determined b o t h spectrofluorometrically and b y the in vitro epidermally mediated covalent binding of DMBA and DBA to DNA. In addition, a dose-response study was undertaken to determine the inhibition of DMBA initiation b y 7,8-BF and the effects of 7,8-BF and 5,6-BF on t u m o r initiation by MC and 7-OHMe12MeBA. MATERIALS AND METHODS
Animals. Female Charles River CD-1 mice were purchased from Charles River Mouse Farms, North Wilmington, Mass. Mice 7 to 9 weeks old were carefully shaved with surgical clippers t w o days before treatment and only those mice in the resting phase of the hair cycle were used in the biochemical and t u m o r experiments. Mice were always killed between 8:00 a.m. and 12:00 p.m. to minimize the effects of diurnal variations. Chemicals. MC was purchased from J.T. Baker, Philipsburg, N.J. and
299 DMBA and DBA from Sigma Chemical Company, St. Louis, Mo. 7,8-BF and 5,6-BF were obtained from Aldrich Chemical Company, Inc., Milwaukee, Wisc. 7-OHMe-12MeBA was a generous gift from Dr. Peter Sims, The Chester Beatty Research Institute, London, Great Britain. [3H] DMBA 99% pure (6.4 Ci/mmole) and [3H]MC 98% pure (16.2 Ci/mmole) were obtained from Amersham/Searle, Arlington Heights, Ill., and [3H] DBA 98% pure (2.42 Ci/ mmole) was purchased from New England Nuclear Corp., Boston, Mass. Tumor induction experiments. Each experimental group contained 30 preshaven mice. DMBA, MC or 7~)HMe-12MeBA in 0.2 ml of acetone was applied topically to the shaved area of the back. 7,8-BF and 5,6-BF were applied topically in 0.2 ml acetone either 6 h prior to, 5 min before or 6 h after initiation with the hydrocarbons. Starting one week after initiation mice received twice weekly applications of 10/~g of TPA in 0.2 ml of acetone. Promotion was continued for 30 weeks and then terminated. The incidence of b o t h papillomas and carcinomas was observed weekly and, at random, papillomas and carcinomas were removed for histological verification. A H H enzyme assay. Three mice were used for each enzyme assay. Mice were killed by cervical dislocation and Nudit cream (supplied by Helena Rubinstein, Inc.) was applied to the shaved area of the back. After 5 min the Nudit cream was thoroughly washed off under cold, running water and the skins were removed and placed on ice. The whole skin was placed dermis side d o w n on a cold glass plate and the epidermis was scraped off with fifteen strokes o f a razor blade and placed in 1 ml of a 0.25 M sucrose--0.05 M Tris buffer, pH 7.5. The epidermal material from three mice was homogenized with a Polytron PT10 homogenizer for 45 sec at setting " 6 " . This epidermal homogenate was the source of enzyme for AHH assays. The assays were performed in semi
Determination of covalent binding of [3H] DMBA, [3H] MC and [3H] DBA
300
to D N A by epidermal homogenates. The assay system for epidermal-homogenate-mediated binding of [3H]DMBA, [3H]MC and [3H]DBA to DNA is based on the system originally described by Gelboin [7] and Grover and Sims [21]. The epidermal material from female Charles River CD-1 mice, that had been pretreated with 200 nmoles of DMBA, MC or DBA for 18 h, was homogenized (5 skins/ml of buffer) as described above and used in the binding studies. The standard reaction mixture contained the following components in a final volume of 3 ml: 50 pmoles sodium phosphate, pH 7.4, 100 pmoles EDTA; 0.5 mg NADPH; 2 mg calf t h y m u s DNA; 0.2 ml of epidermal homogenate (4--6 mg of protein) and 36 nmoles (20 pCi) of either [3H]DMBA, [3H] MC or [3HI DBA in 25 pl of ethanol. In some cases 7,8-BF or 5,6-BF were added in ethanol at the final concentration as stated in the text. The reaction was started by adding the hydrocarbon substrate and the mixture was incubated for 15 min at 37°C in the dark. The reaction was stopped by adding 3 ml of the " s t o p " solution (2% SDS; 0.03 M NaC1; and 0.003 M Na Citrate, pH 7.0). The mixture was extracted twice with 2 vols. of a SDS-saturated phenol solution and nucleic acids were precipitated from the aqueous phase b y the addition of 3 vols. of ice-cold 95% ethanol. The pellet was dissolved in H20 and then 0.3 ml of 2 M MgCl2 was added, followed b y 3 vols. of 95% ethanol. After centrifugation of 2200 rpm for 20 min, the pellet was washed twice more with 95% ethanol followed b y one ether wash. The final nucleic acid pellet was hydrolysed in 0.5 N PCA at 90°C for 20 min and aliquots taken for determination of DNA by the diphenylamine reaction [22] and radioactivity by liquid scintillation counting. The specific activity of binding is expressed as pmoles of hydrocarbon bound/~g o f DNA/mg of protein/15 min of incubation. A zero incubation value was subtracted from each specific activity and each experiment was done in triplicates. In most cases each specific activity represents the average of 2 experiments. RESULTS Dose-response studies on the capacity of 7,8-BF to inhibit t u m o r initiation b y DMBA revealed that 7,8-BF was an effective inhibitor at concentrations equivalent to that of DMBA (2.56 pg), and a m a x i m u m inhibition was observed at 20 times that of DMBA {Fig. 1). Doses of 100 or 1000 pg of 7,8BF had very little addition inhibitory effect on DMBA initiation (Fig. 1). Table I illustrates that DMBA skin t u m o r initiation was inhibited b y 75% with a single application of 25 pg of 7,8-BF; whereas, three doses of 25 pg each of 7,8-BF given 6 h prior to, during and 6 h after DMBA initiation inhibited tumorigenesis b y 87%. Also shown in Table I is the t u m o r initiating ability of 7~)HMe-12MeBA and the effect of 7,8-BF on 7-OHMe-12MeBA initiation. 7,8-BF did n o t inhibit the initiating ability of 7-OHMe-12MeBA and, in fact, slightly increased its initiating action. We also have found that 7,8-BF inhibited MC t u m o r initiation (Fig. 2). Also shown in Fig. 2 is the inhibitory effect of 5,6-BF on MC t u m o r initia-
301 100
5
1 9
13
17 21 25 WEEKS OF PROMOTION
29
Fig. 1. Dose-response relationship for the inhibitory effect of 7,8-BF on DMBA initiation and followed 1 week later by .twice weekly applications of 0.2 ml of 10/.~g of TPA. Each group contained 30 mice, and all mice were initiated with 2.56/2g of DMBA. Those mice that received 7,8-BF were treated 5 rain before initiation. Top: Percentage of mice with papillomas as function of weeks of tumor promotion. Bottom: Average papillomas (Pa)/mouse as function of weeks of tumor promotion. Time measured from first application of promoting agent. DMBA initiation only (o). Dose levels of 7,8-BF were: t , 12.5 pg ( i , 2.7 pg gave similar results); A, 25 pg; o, 50 big; I , 100 pg (1 mg gave similar results).
tion; however, the inhibition was less than that observed for 7,8-BF. (7,8BF and 5,6-BF inhibited MC initiation by 77 and 57%, respectively, 30 weeks after promotion). 7,8-BF has been found to inhibit DMBA and MC initiation of skin tumors and to either stimulate or have no effect on the initiating ability of BP, DBA and 7-OHMe-12MeBA. In general, 5,6-BF slightly inhibited the initiating ability of all the polycyclic hydrocarbons tested. Fig. 3 reveals that epidermal AHH activity was increased by 5,6-BF and had no effect or was slightly inhibited by 7,8-BF when given i.p. at a dose level of 100 or 500/zg. Gelboin et al. [8] reported that an i.p. injection of 7,8-BF induced AHH activity 16 h after treatment; whereas, we consistently had no effect or a slight inhibitory effect from 6 to 12 h after treatment with no significant induction at any time point investigated. We also found that topical application of 7,8-BF inhibited epidermal AHH whereas, 5,6-BF increased epidermal AHH activity as was previously reported [18--20]. Both 7,8-BF and 5,6-BF when added directly to the assay tubes inhibited the in
302 ~L 0
© Cb ~J
0
~J C~
z < ~J
z
.o
o
<
E z 0
© N
e~
< N o
z © z © °~
0 0
r~
Z~
0~.~
~
H
O 0
.~ ~,,'~ ~ ~. <<
~
mmmmm~
-~ .
~'~= ~
~
~ ~.__. ~ o'~
<=z
~J
303 100 u3 <
~Z 0 .J
~: 5o
8
w 6
tD
~4 fL
2
B
12 16 20 24 2• WEEKS OF PROMOTION Fig. 2. The effect of 7,8-BF and 5,6-BF on MC tumor initiation and followed 1 week later by twice weekly applications of 0.2 ml of 10 pg of TPA. Each group contained 30 CD-1 female mice and all mice were initiated with 13.4 pg of MC. 7,8-BF and 5,6-BF were applied topically 5 min before initiation. Top: Percentage of mice with papillomas as function of weeks of tumor promotion; Bottom: Average papillomas (Pa)/mouse as function of weeks of promotion, e, MC initiation only; o, 100 #g of 5,6-BF; A, 100 #g of 7,8-BF.
vitro e p i d e r m a l A H H activity f r o m MC p r e t r e a t e d mice b y greater t h a n 90% (Table II). T h e i n h i b i t i o n o f A H H activity b y b o t h 7,8-BF and 5,6-BF was a little less f r o m c o n t r o l mice. As can b e seen in T a b l e II, p h e n o b a r b i t a l had n o e f f e c t w h e n a d d e d in vitro, as B o w d e n et al. previously r e p o r t e d f o r D M B A p r e t r e a t e d mice [ 2 0 ] . Estradiol was f o u n d t o be a v e r y p o t e n t i~hibit o t o f A H H activity w h e n a d d e d in vitro as was t e s t o s t e r o n e , a l t h o u g h t o a lesser degree. Similar results were r e p o r t e d b y N e b e r t et al. [15] f o r t h e skin and b y B o o t h et al. [23] f o r t h e liver. H o w e v e r , we f o u n d t h a t b o t h estradiol and t e s t o s t e r o n e h a d n o e f f e c t o n D M B A skin t u m o r initiation (Table I). Bates r e p o r t e d [24] t h a t d u r i n g t h e diestrus phase o f t h e cycle, f e m a l e mice are m o r e susceptible t h a n during t h e estrus phase. This finding suggests t h a t estrogens m a y inhibit t h e initiation o f tumorigenesis b y DMBA. We are curr e n t l y d o i n g an extensive dose-response s t u d y w i t h estradiol in o r d e r t o d e t e r m i n e if this discripancy exists at all doses.
304
"-'500 _1
o
I-
~400 L300 )t-r-
2O0
U
IT 100 rl I
6
I
I
I
12 18 24 HOURS AFTER TREATMENT
I
30
I
36
Fig. 3. The e f f e c t o f a single i.p. i n j e c t i o n o f 500 pg ( e ) a n d 100 pg (o) o f 5,6-BF and 500 pg (A) and 100 pg (A) o f 7,8-BF o n A H H activity in m o u s e e p i d e r m i s . T h e specific activity is d e f i n e d as p m o l e s o f 3-OHBP f o r m e d in 30 rain o f i n c u b a t i o n per m g o f p r o t e i n . The average c o n t r o l value was 35 and t h e m a x i m a l l y i n d u c e d value was 210. T h e specific activity is e x p r e s s e d as a p e r c e n t o f t h e c o n t r o l groups and each t i m e p o i n t t e s t e d repr e s e n t s an average o f 3 to 5 g r o u p s c o n t a i n i n g 3 m i c e each. The average p e r c e n t s t a n d a r d d e v i a t i o n f o r t h e c o n t r o l g r o u p s is p l o t t e d o n t h e c h a r t t o i n d i c a t e t h e e x t e n t o f variation in t h e data. T A B L E II IN V I T R O E F F E C T O F B E N Z O F L A V O N E S , P H E N O B A R B I T A L , E S T R A D I O L , A N D T E S T O S T E R O N E ON A H H A C T I V I T Y F R O M M O U S E E P I D E R M I S The m i c e w e r e p r e t r e a t e d topically w i t h 200 n m o l e s o f MC 18 h b e f o r e sacrificing a In vitro a d d i t i o n b
Concentration
% Inhibition c
(M) 7,8-BF 7,8-BF 5,6-BF 5,6-BF Phenobarbital Phenobarbital estradiol estradiol testosterone testosterone
10 - 4 2 x 10 - 4 10 - 4 2 x 10 - 4 10 _ 4 2 x 10 - 4 10 - 4 2 x 10 - 4 10 - 4 2 x 10 - 4
Control
MC-induced
76 88 82 85 2 0
91 95 93 95 5 3 94 96 52 75
a The e n z y m e source for t h e assays was e p i d e r m a l h o m o g e n a t e s and t h e s u b s t r a t e c o n c e n t r a t i o n was 10 -4 M BP. The average specific acitivity for t h e M C - t r e a t e d m i c e was 334 p m o l e s o f 3-OHBP f o r m e d / m g o f p r o t e i n . b The c o m p o u n d s in a c e t o n e w e r e a d d e d d i r e c t l y t o t h e i n c u b a t i o n tubes. c The values in this table are t h e p e r c e n t a g e s o f decrease in specific activity relative to t h e value o b t a i n e d w i t h o u t a d d i n g a m o d i f i e r .
305 TABLE HI THE EF F E C T S OF 7,8-BF, 5,6-BF ESTRADIOL, TESTOSTERONE AND PHENOBARBITAL ON THE IN V I T R O COVALENT BINDING OF T R I T I A T E D DMBA AND DBA BY EPIDERMAL HOMOGENATES a Concentration (nmoles)
In vitro addition b
complete (no addition) 7,8-BF 7,8-BF 5,6BF 5,6-BF estradiol estradiol testosterone testosterone Phenobarbital Phenobarbital
36 72 36 72 36 72 36 72 36 72
Specific activity (× 104) c [3H]DMBA
[3H]DBA
7.57 3.73 2.12 3.58 3.42 4.54 4.02 5.96 5.15 7.68 7.51
1.38 0.49 0.34 0.34 0.21 0.64 0.57 1.10 1.08 1.58 1.48
a The mice were treated topically with 100 nmoles o f either unlabeled DMBA or DBA 18 h before sacrificing. See Materials and Methods for details concerning assay. b 7,8-BF, 5,6-BF, estradiol, testosterone and Phenobarbital were added to the assay tubes at a concentration equivalent to (36 nmoles) or twice the amount (72 nmoles) of DMBA or DBA substrate. c Specific activity is expressed as pmoles bound/p~ of DN.A/mg of protein/15 rain of incubation at 37°C in the dark. TABLE IV THE E F F E C T OF TOPICAL APPLICATION OF 100 btg OF 7,8-BF AND 2.56 pg OF DMBA ON THE IN VITRO COVALENT BINDING OF T R I T I A T E D DMBA TO DNA BY EPIDERMAL HOMOGENATES FROM THE P R E T R E A T E D MICE a Time after pretreatment
Pretreatment
Spec. act. (× 104) b
Spec. act. (% of controls) c
DMBA DMBA + 7,8-BF DMBA DMBA+ 7,8-BF DMBA DMBA+ 7,8-BF DMBA DMBA+ 7,8-BF DMBA DMBA+ 7,8-BF
4.48 d 5.10 6.21 3.54 8.29 5.14 3.91 4.03 4.11 6.86
100 114 100 57 100 62 100 103 100 167
(h) 1 1 3 3 6 6 12 12 24 24
a The mice were treated topically with 100/~g of 7,8-BF 5 rain before unlabeled DMBA (2.55 #g) and sacrificed at the times indicated. See Materials and Methods for details concerning assay. b Specific activity (Spec. act.) ix expressed as pmoles bound//~g DNA/mg of protein/15 rain of incugation at 37°C in the dark. c Specific activity (Spec. act.) is expressed as percentage of THE DMBA control. d Each value represents 2 experiments with duplicate determinations per experiment.
306 0 ¢i,
0
L
o ~
0
rj
U v
i
~'~
~o
Z o N
~-/
~% %, t'-
"r! '
,~,~
.~
307 Table III notes the effects of adding several AHH modifying agents on the in vitro covalent binding of tritiated DMBA and DBA to DNA b y epidermal homogenates. When added directly to the assay, 7,8-BF and 5,6-BF inhibited the epidermally-mediated covalent binding of [aH] DMBA and [3H] DBA to DNA. Although n o t shown in Table III, 7,8-BF and 5,6-BF had a similar inhibitory effect on the epidermally-mediated convalent binding of [all] MC and [3H] BP to DNA when added directly to the assay tubes. Estradiol and testosterone also inhibited the in vitro binding b u t to a lesser degree than the benzoflavones. Phenobarbital had no effect on AHH activity (Table II) and also had no effect on DMBA and DBA covalent binding to DNA b y epidermal homogenates. Similar binding results were obtained using epidermal microsomes but, because of the large number of mice needed per assay, we decided to use the epidermal homogenates. The in vitro covalent binding of tritiated DMBA to DNA b y epidermal homogenate from 7,8-BF and/or DMBA pretreated mice is shown in Table IV. The specific activity of binding of [3H]DMBA to DNA b y epidermal homogenates in vitro, as a function of time after induction with 2.56/~g of unlabeled DMBA in vivo revealed that the specific activity of binding reached a peak by 6 hours. As can be seen, 7,8-BF was effective in decreasing this in vitro binding only at 3 and 6 hours after pretreatment to the mice. The epidermal homogenates from mice pretreated with 7,8-BF and DMBA 24 h before sacrificing brought a b o u t a high level of in vitro binding than
TABLE
V
T H E E F F E C T O F T O P I C A L A P P L I C A T I O N O F 100 pg O F 7,8-BF A N D 13.4 pg O F M C O N T H E IN V I T R O C O V A L E N T BINDING OF [3H]MC TO DNA BY EPIDERMAL HOMOGENATES FROM THE PRETREATED MICE a Time after p r e treatment (h)
Pretreatment
Spec. act. (× 104 )b
Spec. act. (% of controls) c
1 1 3
MC MC + 7,8-BF MC
1.45 d 1.12 1.62
I00 77 100
3 6
MC + 7,8-BF MC M C + 7,8-BF MC
1.23 4.44 2.95
76 100 66
5.85 5.56 3.74 3.98
1O0 95 100 106
6
12 12 24 24
MC + 7,8-BF MC MC + 7,8-BF
a The mice were treated topically with 100 pg of 7,8-BF 5 rain before unlabeled M C (13.4 pg) and sacrificed at the times indicated. See Materials and Methods for details concerning the in vitro assay. b Specific activity (Spec. act.) is expressed as pmoles bound per pg D N A per m g of protein per 15 rain of incubation at 37°C in the dark. c Specific activity (Spec. act.) is expressed as percentage of the M C control. d Each value represents 2 experiments with duplicate determinations per experiment.
308 TABLE VI T H E E F F E C T O F T O P I C A L A P P L I C A T I O N O F 1 0 0 ~g O F 7 , 8 - B F A N D 56 gg D B A ON T H E IN V I T R O C O V A L E N T B I N D I N G O F [ 3 H ] D B A T O D N A BY E P I D E R M A L H O M O G E N A T E S F R O M T H E P R E T R E A T E D MICE a T i m e a f t e r pret r e a t m e n t (h) 1 1 3 3 6 6
12 12 24 24
Pretreatment
Spec. act. (x 104 ) b
Spec. act. (% of c o n t r o l s ) c
DBA D B A + 7,8-BF DBA D B A + 7,8-BF DBA DBA + 7,8-BF DBA D B A + 7,8-BF DBA D B A + 7,8-BF
1.12 d 1.20 1.23 1.28 2.41 2.46 2.62 2.94 2.10 2.85
100 107 100 104 100 102 100 112 100 136
a T h e m i c e were t r e a t e d t o p i c a l l y w i t h 1 0 0 /~g o f 7 , 8 - B F 5 rain b e f o r e u n l a b e l e d D B A (56 pg) a n d sacrificed a t t h e t i m e s i n d i c a t e d . See Materials a n d M e t h o d s for details conc e r n i n g t h e in v i t r o assay. b Specific a c t i v i t y (Spec. a c t . ) is e x p r e s s e d as p m o l e s b o u n d p e r pg D N A p e r m g o f prot e i n p e r 15 m i n o f i n c u b a t i o n at 3 7 ° C in t h e d a r k . c Specific a c t i v i t y (Spec. act. ) is e x p r e s s e d as p e r c e n t a g e o f t h e D B A c o n t o l . d E a c h value r e p r e s e n t s 2 e x p e r i m e n t s w i t h d u p l i c a t e d e t e r m i n a t i o n s p e r e x p e r i m e n t .
only DMBA pretreatment. In experiments similar to those depicted in Table IV, the in vitro covalent binding of [3H] MC to DNA by epidermal homogenates was also inhibited by early (1--6 h) pretreatment with 7,8-BF and MC as compared to only MC (Table V). However, the in vitro covalent binding of [3HI DBA to DNA by epidermal homogenates was either not significantly changed or was slightly increased by 7,8-BF and DBA pretreatment as compared to DBA only (Table VI). DISCUSSION
Several chemical carcinogens have been shown to bind covalently to cellular nucleic acids and proteins [ 3,6,25--27], and it seems likely that the interaction with one or more of these macromolecules is essential for the carcinogenic process. Previous studies from this laboratory have demonstrated that a correlation exists between tumor initiating ability of several polycyclic hydrocarbons and their ability to bind covalently to DNA in vitro upon incubation with epidermal homogenates and NADPH as the electrophile-generating system [25]. This is in agreement with early reports which showed that a correlation was found between the extent of in vivo binding of several polycyclic hydrocarbons to mouse skin DNA [17] and protein [16] and the carcinogenic activity of the hydrocarbons. In fact, the Millers [3,28] have proposed a significant general theory to explain chemical carcinogenesis.
309 Their theory states that all chemical carcinogens that are not electrophilic reactants must be converted metabolically into a chemically reactive electrophilic form. From the above one would conclude that if the convalent binding of carcinogenic polycyclic hydrocarbons to some critical nucleophile were decreased its carcinogenic activity also would be decreased. In general, this seems to be an accurate deduction, although there are a few situations where the phenomenon becomes unclear. For example, when 7,8-BF and 5,6-BF are added directly to the incubation tubes, they inhibit the epidermally mediated covalent binding of [3HI DMBA and [3H] DBA to DNA. Similar results recently were noted in this laboratory when [3H]BP and [3H]MC were used [29]. Furthermore, Selkirk et al. [30] demonstrated 7,8-BF inhibited the formation of hydroxylated products from BP by rat liver microsome preparations. In addition, experiments in our laboratory using HPLC indicated that when both 7,8-BF and 5,6-BF were added in vitro they inhibited the formation of all BP and DMBA metabolites by rat liver and mouse epidermal microsomes [29,31]. However, as stated earlier our results plus those of Bowden et al. [20] and Kinoshita and Gelboin [9] showed that 7,8BF inhibited the tumor initiating ability of DMBA and MC but either had no effect or enhanced the initiating ability of BP and DBA. 5,6-BF, in general, exhibited a slight inhibitory effect on the tumor initiation of all the hydrocarbons tested [9,18,20]. As shown in Tables IV, V and VI, a correlation was observed between the effects of 7,8-BF on various PAH initiated tumors and the in vitro covalent binding of these PAH to DNA. This relationship was noted when the binding assays consisted of epidermal homogenates from mice treated in the same manner as those used in the tumor initiation experiments. Epidermal homogenates from mice pretreated with both 7,8-BF and either DMBA or MC (1--6 h before sacrificing) had a lesser capability of converting either [3HI MC or [3H] DMBA to a DNA binding product than mice treated with only DMBA or MC. Whereas, the in vitro binding of DBA to DNA was either unchanged or slightly increases when using epidermal homogenates from mice treated with 7,8-BF and DBA as compared to only DBA treated mice. This was true at all time points investigated (Table VI). Recent results reported by Sloane et al. [32--34] may help clarify the somewhat contradictory results reported on the effects of 7,8-BF on PAH carcinogenesis. They demonstrated that model aromatic compounds, including BP, could be metabolized to the aryl hydroxymethyl derivative by either direct hydroxymethylation (independent of cytochrome P-450 dependent mixed function oxidation) or direct oxidation of the aryl side-chain methyl. Also, Sloane [34] found that 7,8-BF activates the aryl hydroxymethyl synthetase of rat liver and lung which catalyzed the hydroxymethylation of BP. Conversely, 5,6-BF had no effect on the synthesis enzyme system [26]. Flesher and Sydnor [35] and Dewhurst et al. [36] reported that 6-hydroxymethyl-BP was a carcinogen. Its tumor initiating ability in mouse skin is currently being investigated.
310 A p r o p o s e d m e t a b o l i c s c h e m e f o r the cellular m e t a b o l i s m o f P A H w h i c h c o u l d a c c o u n t f o r t h e effects o f 7,8-BF and 5,6-BF o n P A H carcinogenesis in m o u s e skin is s h o w n in Fig. 4. A c t i v a t i o n o f P A H to p r o x i m a t e a n d / o r u l t i m a t e c a r c i n o g e n s is r e p r e s e n t e d b y 3 p a t h w a y s . T w o p a t h w a y s , B and C involve o x i d a t i o n and t h e third, A, does n o t involve o x i d a t i o n o f the P A H b y c y t o c h r o m e P-450 d e p e n d e n t o x y g e n a s e s . I f o n e p o s t u l a t e s t h a t the m e t h y l a t e d P A H , D M B A a n d MC, are n o t e f f e c t i v e l y m e t a b o l i z e d via pathw a y A ( h y d r o x y m e t h y l a t i o n ) because o f the presence o f the m e t h y l g r o u p , t h e n a m e c h a n i s m can be p r o p o s e d f o r the m o d i f i c a t i o n o f skin t u m o r i g e n e sis b y 7,8-BF and 5,6-BF. 7,8-BF h a d e i t h e r n o e f f e c t or e n h a n c e s BP a n d D B A skin t u m o r initiation b y partially inhibiting t h e i r o x i d a t i o n b y p a t h w a y C and activating p a t h w a y A. As stated earlier [ 3 4 ] 5,6-BF has n o e f f e c t o n p a t h w a y A ( h y d r o x y m e t h y l a t i o n ) . 7,8-BF drastically c o u n t e r a c t s D M B A and MC skin t u m o r initiation b y p r e v e n t i n g their o x i d a t i o n t o either an e p o x i d e a n d / o r activated m e t h y l g r o u p . 5,6-BF m o d e r a t e l y inhibits D M B A , MC, D B A and BP skin t u m o r initiation b y a similar m e c h a n i s m b u t the differences in m a g n i t u d e o f t h a t i n h i b i t i o n m a y be related to t h e fact t h a t 7,8-BF does n o t i n d u c e e p i d e r m a l A H H activity whereas 5,6-BF does. REFERENCES 1 A.H. Conney, Pharmacological implication of microsomal enzyme induction Pharmacol. REv., 19 (1967) 317. 2 H.V. Gelboin, Carcinogens, enzyme induction, and gene activation, Adv. Cancer Res., 10 (1967) 1. 3 E.C. Miller and J.A. Miller, Biochemical mechanisms of chemical carcinogenesis in the molecular biology of cancer, in H. Busch (Ed.), Academic Press, New York, 1974, pp. 377--402. 4 E. Boyland, The biological significance of metabolism of polycyclic compounds, Symp. Biochem. Soc., 5 (1950) 40. 5 P. Sims, The metabolism of benzo(a)pyrene by rat liver homogenates, Biochem. Pharmacol., 16 (1967) 613. 6 C. Heidelberger, Chemical carcinogenesis, Annu. REv. Biochem., 44 (1975) 79. 7 H.V. Gelboin, A microsome-dependent binding of benzo(a)pyrene to DNA, Cancer Res., 29 (1969) 1272. 8 H.V. Gelboin, F.J. Wiebel and L. Diamond, Dimethylbenzanthracene tumorigenesis and aryl hydrocarbon hydroxylase in mouse skin. Inhibition by 7,8-benzoflavone, Science, 170 (1970) 169. 9 H.B. Gelboin and F.J. Wiebel, Studies on the mechanism of aryl hydrocarbon hydroxylase induction and its role in cytotoxicity and tumorigenesis, Ann. N.Y. Acad. Sci., 170 (1971) 529. 10 K. Burki, A.G. Liebelt and E. Bresnick, Induction of aryl hydrocarbon hydroxylase in mouse tissues from a high and low cancer strain and their F1 hybrids, J. Natl. Cancer Inst., 50 (1973) 369. 11 S. ThompsOn and T.J. Slaga, Mouse epidermal aryl hydrocarbon hydroxylase, J. Invest. Dermatol., 66 (1976) 108. 12 F.J. Wiebel, B.S. Leutz and H.V. Gelboin, Aryl hydrocarbon (benzo(a)pyrene)hydroxylase: A mixed-function oxygenase in mouse, J. Invest. Dermatol., 64 (1975) 184. 13 R.J. Pohl, R.M. Philpot and J.R. Fouts, Cytochome P-450 content and mixed-func-
311
14 15
16
17 18
19
20
21 22 23
24
25
26
27
28 29
30
31
32
tion oxidase activity in microsomes isolated from mouse skin, Drug Metab. Dispos., 4 (1976) 442. A.P. Poland, E. Glover, J.R. Robinson and D.W. Neberg, Genetic expression of aryl hydrocarbon hydroxylase activity 249 (1974) 5599. D.W. Nebert, L.L. Bausserman and R.'R. Bates, Effect of 17~-estradiol and testosterone on aryl hydrocarbon hydroxylase activity in mouse tissues in vivo and in cell culture, Int. J. Cancer, 6 (1970) 470. L.W. Wattenberg and J.L. Leong, Inhibition of the carcinogenic action of 7,12-dimethylbenz(a)anthracene by ~-naphthoflavone, Proc. Soc. Exp. Biol. IVied., 128 (1968) 940. L.W. Wattenberg and J.L. Leong, Inhibition of the carcinogenic action of benzo(a)pyrene by flavones. Cancer Res., 30 (1970) 1922. N. Kinoshita and H.V. Gelboin, The role of aryl hydrocarbon hydroxylase in 7,12dimethylbenz(a)anthracene skin tumorigenesis: on the mechanism of 7,8-benzoflavone inhibition of tumorigenesis, Cancer Res., 32 (1972) 1329. N. Kinoshita and H.V. Gelboin, Aryl hydrocarbon hydroxylase and polycyclic hydrocarbon tumorigenesis: Effect of the enzyme inhibitor 7,8-benzoflavone in tumorigenesis and macromolecular binding, Proc. Natl. Acad. Sci. U.S., 69 (1972) 824. G.T. Bowden, T,J. Slaga, B.G. Shapas and R.K. Boutwell, The role of aryl hydrocarbon hydroxylase in skin tumor initiation by 7,12-dimethylbenz(a)anthracene and 1,2,5,6-dibenzanthracene using DNA binding and thymidine-3H incorporation into DNA as criteria, Cancer Res., 34 (1974) 2634. P.L. Grover and P. Sims, Enzyme-catalyzed reactions of polycyclic hydrocarbons with DNA and protein in vitro,Biochem. J., 110 (1968) 159. K.A.. Burton, Study of the conditions and mechanism of the diphenylamine reaction for the colorimeter estimation of DNA, Biochem. J., 62 (1965) 315. J. Booth, G.R. Keysell and P. Sims, Effects of oestradiol on the in vitro metabolism of 7,12-dimethylbenz(a)-anthracene and its hydroxymethyl derivatives. Biochem. Pharmacol., 23 (1974) 735. R.R. Bates, Sex hormones and skin tumorigenesis. I. Effect of the estrus cycle and castration on tumorigenesis by 7,12-dimethyl(a)anthracene. J. Natl. Cancer Inst., 41 (1968) 559. S.G. Bury, S. Thompson and T.J. Slaga, The role of epidermal aryl hydrocarbon hydroxylase in the covalent binding of polycyclic hydrocarbons to DNA and its relationship to tumor initiation. Biochem. Biophys. Res. Commun., 70 (1976) 1102. C. Heidelberger and M.G. Moldenhauer, The interaction of carcinogenic hydrocarbons with tissue constituents. IV. A quantitative study of the binding to skin proteins of several C 14-labeled hydrocarbons. Cancer Res., 16 (1956) 442. P. Brookes and P.D. Lawley, Evidence for the binding of polynuclear aromatic hydrocarbons to the nucleic acid of mouse skin: relation between carcinogenic power of hydrocarbons and their b i n d i n g t o deoxyribonucleic acid. Nature, 202 (1964) 781. J.A. Miller, Carcinogenesis by chemicals: An overview. G.H.A. Clowes Memorial Lecture. Cancer Res., 30 (1970) 559. T.J. Slaga, D.L. Berry, M.R. Juchau, S. Thompson, S.G. Buty and A. Viaje, Effects of benzoflavones and trichloropropene oxide on polynuclear aromatic hydrocarbon metabolism and initiation of skin tumors in polynuclear aromatic hydrocarbons, in R.I. Freudenthal and D.W. Jones (Eds.), Raven Press, New York, 1976, pp. 127--137. J.K. Selkirk, R.G. Croy, P.P. Roller and H.V. Gelboin, High-pressure liquid chromatographic analysis of benzo(a)pyrene metabolism and covalent binding and the mechanism of action of 7,8-benzoflavone and 1,2-epoxy-3,3,3-trichloropropane, Cancer Res.., 34 (1974) 3474. J. DiGiovanni, T.J. Slaga, D.L. Berry and M.R. Juchau, Metabolism of 7,12-dimethylbenzanthracene in mouse skin homogenates analyzed with high-pressure liquid chromatography. Drug Metab. Dispos., (1977 ) In press. N.H. SIoane and T.K. Davis, Hydroxymethylation of the benzene ring. Microsomal
312 hydroxymethylation of benzo(a)pyrene, Arch. Biochem. Biophys., 163 (1974) 46. 33 N.H. Sloane and M. Heinemann, Hydroxymethylation of the benzene ring. V. NADPH requirement for aryl hydrooxymethylation by a C-1 donor b o u n d to a macromolecule, Biochim. Biophys. Acta., 201 (1970) 384. 34 N.H. Sloane, ~-Naphthoflavone activation of 6-hydroxymethylbenzo(a)pyrene synthetase, 35 (1975) 3731. 35 J.W. Flesher and K.L. Sydnor, Possible role of 6-hydroxymethylbenzo(a)pyrene as a proximate carcinogen of benzo(a)pyrene and 6-methylbenzo(a)pyrene, Int. J. Cancer, 11 (1973) 433. 36 F. Dewhurst, D.A. Kitchen and G. Calcutt, The carcinogenicity of some6-substituted benzo(a)pyrene derivatives in mice. Br. J. Cancer 26 (1972) 506.
Chem.-Biol. Interactions, 17 (1977) 297--312 © Elsevier/North-Holland Scientific Publishers, Ltd.
THE EFFECTS OF BENZOFLAVONES ON POLYCYCLIC HYDROCARBON METABOLISM AND SKIN TUMOR INITIATION
*
THOMAS J. SLAGA, SARA THOMPSON **, DAVID L. BERRY, JOHN DIGIOVANNI, MONT R. JUCHAU and AURORA VIAJE
Cancer and Toxicology Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 3 7830 and Department of Pharmacology, University of Washington, School of Medicine, Seattle, Wash. 98195 (U.S.A.) (Received October 28th, 1976) (Revision received February 4th, 1977) (Accepted February 28th, 1977)
SUMMARY
The effects of benzoflavones on skin tumor initiation by polycyclic hydrocarbons and epidermal aryl hydrocarbon hydroxylase were investigated. 7,8-Benzoflavone (7,8-BF) was found to be a potent inhibitor of the inhibition of skin tumors by 3-methylcholanthrene (MC) as well as 7,12
298 to be partially related to its ability to inhibit the formation of electrophilic intermediates.
INTRODUCTION
The microsomal enzyme complex of mixed function oxidases is responsible for the detoxification of a variety of compounds, including polycyclic aromatic hydrocarbons [1--6]. Gelboin et al. [7--9] have also proposed that the specific enzyme complex AHH might be responsible for the activation of polycyclic hydrocarbons to toxic and carcinogenic metabolites. This enzyme complex has been found in most mammalian tissues including mouse skin in which it is highly inducible [8,10--15]. Wattenberg and Leong [16,17] reported that i.p. injection of various benzoflavones inhibited polycyclic hydrocarbon induced tumorigenesis in the lung, mammary gland and intestine of rats and mice. 7,8-BF given topically has been shown to inhibit skin t u m o r initiation b y DMBA b u t to enhance t u m o r initiation by BP [8,18--20]. This flavone inhibited the binding of DMBA to DNA, R N A and protein and, to a lesser degree, BP binding to macromolecules [19]. Bowden et al. [20] reported that 7,8-BF inhibited DMBA skin t u m o r initiation, decreased DMBA binding to epidermal macromolecules and reversed the DMBA inhibition of DNA synthesis. They also reported that 7,8-BF stimulated tumorigenesis with DBA and had no effect on the covalent binding of DBA to macromolecules. 5,6-BF slightly inhibited b o t h t u m o r formation and hydrocarbon binding in the case of either DMBA or DBA [20]. The above data led Bowden et al. [20] to hypothesize that methylated polycyclic hydrocarbons are metabolized in a different manner from non-methylated ones b y the epidermal A H H system, especially under the influence of 7,8-BF. In an effort to better understand this relationship, the effects of 7,8-BF and 5,6-BF on epidermal A H H were determined b o t h spectrofluorometrically and b y the in vitro epidermally mediated covalent binding of DMBA and DBA to DNA. In addition, a dose-response study was undertaken to determine the inhibition of DMBA initiation b y 7,8-BF and the effects of 7,8-BF and 5,6-BF on t u m o r initiation by MC and 7-OHMe12MeBA. MATERIALS AND METHODS
Animals. Female Charles River CD-1 mice were purchased from Charles River Mouse Farms, North Wilmington, Mass. Mice 7 to 9 weeks old were carefully shaved with surgical clippers t w o days before treatment and only those mice in the resting phase of the hair cycle were used in the biochemical and t u m o r experiments. Mice were always killed between 8:00 a.m. and 12:00 p.m. to minimize the effects of diurnal variations. Chemicals. MC was purchased from J.T. Baker, Philipsburg, N.J. and
299 DMBA and DBA from Sigma Chemical Company, St. Louis, Mo. 7,8-BF and 5,6-BF were obtained from Aldrich Chemical Company, Inc., Milwaukee, Wisc. 7-OHMe-12MeBA was a generous gift from Dr. Peter Sims, The Chester Beatty Research Institute, London, Great Britain. [3H] DMBA 99% pure (6.4 Ci/mmole) and [3H]MC 98% pure (16.2 Ci/mmole) were obtained from Amersham/Searle, Arlington Heights, Ill., and [3H] DBA 98% pure (2.42 Ci/ mmole) was purchased from New England Nuclear Corp., Boston, Mass. Tumor induction experiments. Each experimental group contained 30 preshaven mice. DMBA, MC or 7~)HMe-12MeBA in 0.2 ml of acetone was applied topically to the shaved area of the back. 7,8-BF and 5,6-BF were applied topically in 0.2 ml acetone either 6 h prior to, 5 min before or 6 h after initiation with the hydrocarbons. Starting one week after initiation mice received twice weekly applications of 10/~g of TPA in 0.2 ml of acetone. Promotion was continued for 30 weeks and then terminated. The incidence of b o t h papillomas and carcinomas was observed weekly and, at random, papillomas and carcinomas were removed for histological verification. A H H enzyme assay. Three mice were used for each enzyme assay. Mice were killed by cervical dislocation and Nudit cream (supplied by Helena Rubinstein, Inc.) was applied to the shaved area of the back. After 5 min the Nudit cream was thoroughly washed off under cold, running water and the skins were removed and placed on ice. The whole skin was placed dermis side d o w n on a cold glass plate and the epidermis was scraped off with fifteen strokes o f a razor blade and placed in 1 ml of a 0.25 M sucrose--0.05 M Tris buffer, pH 7.5. The epidermal material from three mice was homogenized with a Polytron PT10 homogenizer for 45 sec at setting " 6 " . This epidermal homogenate was the source of enzyme for AHH assays. The assays were performed in semi
Determination of covalent binding of [3H] DMBA, [3H] MC and [3H] DBA
300
to D N A by epidermal homogenates. The assay system for epidermal-homogenate-mediated binding of [3H]DMBA, [3H]MC and [3H]DBA to DNA is based on the system originally described by Gelboin [7] and Grover and Sims [21]. The epidermal material from female Charles River CD-1 mice, that had been pretreated with 200 nmoles of DMBA, MC or DBA for 18 h, was homogenized (5 skins/ml of buffer) as described above and used in the binding studies. The standard reaction mixture contained the following components in a final volume of 3 ml: 50 pmoles sodium phosphate, pH 7.4, 100 pmoles EDTA; 0.5 mg NADPH; 2 mg calf t h y m u s DNA; 0.2 ml of epidermal homogenate (4--6 mg of protein) and 36 nmoles (20 pCi) of either [3H]DMBA, [3H] MC or [3HI DBA in 25 pl of ethanol. In some cases 7,8-BF or 5,6-BF were added in ethanol at the final concentration as stated in the text. The reaction was started by adding the hydrocarbon substrate and the mixture was incubated for 15 min at 37°C in the dark. The reaction was stopped by adding 3 ml of the " s t o p " solution (2% SDS; 0.03 M NaC1; and 0.003 M Na Citrate, pH 7.0). The mixture was extracted twice with 2 vols. of a SDS-saturated phenol solution and nucleic acids were precipitated from the aqueous phase b y the addition of 3 vols. of ice-cold 95% ethanol. The pellet was dissolved in H20 and then 0.3 ml of 2 M MgCl2 was added, followed b y 3 vols. of 95% ethanol. After centrifugation of 2200 rpm for 20 min, the pellet was washed twice more with 95% ethanol followed b y one ether wash. The final nucleic acid pellet was hydrolysed in 0.5 N PCA at 90°C for 20 min and aliquots taken for determination of DNA by the diphenylamine reaction [22] and radioactivity by liquid scintillation counting. The specific activity of binding is expressed as pmoles of hydrocarbon bound/~g o f DNA/mg of protein/15 min of incubation. A zero incubation value was subtracted from each specific activity and each experiment was done in triplicates. In most cases each specific activity represents the average of 2 experiments. RESULTS Dose-response studies on the capacity of 7,8-BF to inhibit t u m o r initiation b y DMBA revealed that 7,8-BF was an effective inhibitor at concentrations equivalent to that of DMBA (2.56 pg), and a m a x i m u m inhibition was observed at 20 times that of DMBA {Fig. 1). Doses of 100 or 1000 pg of 7,8BF had very little addition inhibitory effect on DMBA initiation (Fig. 1). Table I illustrates that DMBA skin t u m o r initiation was inhibited b y 75% with a single application of 25 pg of 7,8-BF; whereas, three doses of 25 pg each of 7,8-BF given 6 h prior to, during and 6 h after DMBA initiation inhibited tumorigenesis b y 87%. Also shown in Table I is the t u m o r initiating ability of 7~)HMe-12MeBA and the effect of 7,8-BF on 7-OHMe-12MeBA initiation. 7,8-BF did n o t inhibit the initiating ability of 7-OHMe-12MeBA and, in fact, slightly increased its initiating action. We also have found that 7,8-BF inhibited MC t u m o r initiation (Fig. 2). Also shown in Fig. 2 is the inhibitory effect of 5,6-BF on MC t u m o r initia-
301 100
5
1 9
13
17 21 25 WEEKS OF PROMOTION
29
Fig. 1. Dose-response relationship for the inhibitory effect of 7,8-BF on DMBA initiation and followed 1 week later by .twice weekly applications of 0.2 ml of 10/.~g of TPA. Each group contained 30 mice, and all mice were initiated with 2.56/2g of DMBA. Those mice that received 7,8-BF were treated 5 rain before initiation. Top: Percentage of mice with papillomas as function of weeks of tumor promotion. Bottom: Average papillomas (Pa)/mouse as function of weeks of tumor promotion. Time measured from first application of promoting agent. DMBA initiation only (o). Dose levels of 7,8-BF were: t , 12.5 pg ( i , 2.7 pg gave similar results); A, 25 pg; o, 50 big; I , 100 pg (1 mg gave similar results).
tion; however, the inhibition was less than that observed for 7,8-BF. (7,8BF and 5,6-BF inhibited MC initiation by 77 and 57%, respectively, 30 weeks after promotion). 7,8-BF has been found to inhibit DMBA and MC initiation of skin tumors and to either stimulate or have no effect on the initiating ability of BP, DBA and 7-OHMe-12MeBA. In general, 5,6-BF slightly inhibited the initiating ability of all the polycyclic hydrocarbons tested. Fig. 3 reveals that epidermal AHH activity was increased by 5,6-BF and had no effect or was slightly inhibited by 7,8-BF when given i.p. at a dose level of 100 or 500/zg. Gelboin et al. [8] reported that an i.p. injection of 7,8-BF induced AHH activity 16 h after treatment; whereas, we consistently had no effect or a slight inhibitory effect from 6 to 12 h after treatment with no significant induction at any time point investigated. We also found that topical application of 7,8-BF inhibited epidermal AHH whereas, 5,6-BF increased epidermal AHH activity as was previously reported [18--20]. Both 7,8-BF and 5,6-BF when added directly to the assay tubes inhibited the in
302 ~L 0
© Cb ~J
0
~J C~
z < ~J
z
.o
o
<
E z 0
© N
e~
< N o
z © z © °~
0 0
r~
Z~
0~.~
~
H
O 0
.~ ~,,'~ ~ ~. <<
~
mmmmm~
-~ .
~'~= ~
~
~ ~.__. ~ o'~
<=z
~J
303 100 u3 <
~Z 0 .J
~: 5o
8
w 6
tD
~4 fL
2
B
12 16 20 24 2• WEEKS OF PROMOTION Fig. 2. The effect of 7,8-BF and 5,6-BF on MC tumor initiation and followed 1 week later by twice weekly applications of 0.2 ml of 10 pg of TPA. Each group contained 30 CD-1 female mice and all mice were initiated with 13.4 pg of MC. 7,8-BF and 5,6-BF were applied topically 5 min before initiation. Top: Percentage of mice with papillomas as function of weeks of tumor promotion; Bottom: Average papillomas (Pa)/mouse as function of weeks of promotion, e, MC initiation only; o, 100 #g of 5,6-BF; A, 100 #g of 7,8-BF.
vitro e p i d e r m a l A H H activity f r o m MC p r e t r e a t e d mice b y greater t h a n 90% (Table II). T h e i n h i b i t i o n o f A H H activity b y b o t h 7,8-BF and 5,6-BF was a little less f r o m c o n t r o l mice. As can b e seen in T a b l e II, p h e n o b a r b i t a l had n o e f f e c t w h e n a d d e d in vitro, as B o w d e n et al. previously r e p o r t e d f o r D M B A p r e t r e a t e d mice [ 2 0 ] . Estradiol was f o u n d t o be a v e r y p o t e n t i~hibit o t o f A H H activity w h e n a d d e d in vitro as was t e s t o s t e r o n e , a l t h o u g h t o a lesser degree. Similar results were r e p o r t e d b y N e b e r t et al. [15] f o r t h e skin and b y B o o t h et al. [23] f o r t h e liver. H o w e v e r , we f o u n d t h a t b o t h estradiol and t e s t o s t e r o n e h a d n o e f f e c t o n D M B A skin t u m o r initiation (Table I). Bates r e p o r t e d [24] t h a t d u r i n g t h e diestrus phase o f t h e cycle, f e m a l e mice are m o r e susceptible t h a n during t h e estrus phase. This finding suggests t h a t estrogens m a y inhibit t h e initiation o f tumorigenesis b y DMBA. We are curr e n t l y d o i n g an extensive dose-response s t u d y w i t h estradiol in o r d e r t o d e t e r m i n e if this discripancy exists at all doses.
304
"-'500 _1
o
I-
~400 L300 )t-r-
2O0
U
IT 100 rl I
6
I
I
I
12 18 24 HOURS AFTER TREATMENT
I
30
I
36
Fig. 3. The e f f e c t o f a single i.p. i n j e c t i o n o f 500 pg ( e ) a n d 100 pg (o) o f 5,6-BF and 500 pg (A) and 100 pg (A) o f 7,8-BF o n A H H activity in m o u s e e p i d e r m i s . T h e specific activity is d e f i n e d as p m o l e s o f 3-OHBP f o r m e d in 30 rain o f i n c u b a t i o n per m g o f p r o t e i n . The average c o n t r o l value was 35 and t h e m a x i m a l l y i n d u c e d value was 210. T h e specific activity is e x p r e s s e d as a p e r c e n t o f t h e c o n t r o l groups and each t i m e p o i n t t e s t e d repr e s e n t s an average o f 3 to 5 g r o u p s c o n t a i n i n g 3 m i c e each. The average p e r c e n t s t a n d a r d d e v i a t i o n f o r t h e c o n t r o l g r o u p s is p l o t t e d o n t h e c h a r t t o i n d i c a t e t h e e x t e n t o f variation in t h e data. T A B L E II IN V I T R O E F F E C T O F B E N Z O F L A V O N E S , P H E N O B A R B I T A L , E S T R A D I O L , A N D T E S T O S T E R O N E ON A H H A C T I V I T Y F R O M M O U S E E P I D E R M I S The m i c e w e r e p r e t r e a t e d topically w i t h 200 n m o l e s o f MC 18 h b e f o r e sacrificing a In vitro a d d i t i o n b
Concentration
% Inhibition c
(M) 7,8-BF 7,8-BF 5,6-BF 5,6-BF Phenobarbital Phenobarbital estradiol estradiol testosterone testosterone
10 - 4 2 x 10 - 4 10 - 4 2 x 10 - 4 10 _ 4 2 x 10 - 4 10 - 4 2 x 10 - 4 10 - 4 2 x 10 - 4
Control
MC-induced
76 88 82 85 2 0
91 95 93 95 5 3 94 96 52 75
a The e n z y m e source for t h e assays was e p i d e r m a l h o m o g e n a t e s and t h e s u b s t r a t e c o n c e n t r a t i o n was 10 -4 M BP. The average specific acitivity for t h e M C - t r e a t e d m i c e was 334 p m o l e s o f 3-OHBP f o r m e d / m g o f p r o t e i n . b The c o m p o u n d s in a c e t o n e w e r e a d d e d d i r e c t l y t o t h e i n c u b a t i o n tubes. c The values in this table are t h e p e r c e n t a g e s o f decrease in specific activity relative to t h e value o b t a i n e d w i t h o u t a d d i n g a m o d i f i e r .
305 TABLE HI THE EF F E C T S OF 7,8-BF, 5,6-BF ESTRADIOL, TESTOSTERONE AND PHENOBARBITAL ON THE IN V I T R O COVALENT BINDING OF T R I T I A T E D DMBA AND DBA BY EPIDERMAL HOMOGENATES a Concentration (nmoles)
In vitro addition b
complete (no addition) 7,8-BF 7,8-BF 5,6BF 5,6-BF estradiol estradiol testosterone testosterone Phenobarbital Phenobarbital
36 72 36 72 36 72 36 72 36 72
Specific activity (× 104) c [3H]DMBA
[3H]DBA
7.57 3.73 2.12 3.58 3.42 4.54 4.02 5.96 5.15 7.68 7.51
1.38 0.49 0.34 0.34 0.21 0.64 0.57 1.10 1.08 1.58 1.48
a The mice were treated topically with 100 nmoles o f either unlabeled DMBA or DBA 18 h before sacrificing. See Materials and Methods for details concerning assay. b 7,8-BF, 5,6-BF, estradiol, testosterone and Phenobarbital were added to the assay tubes at a concentration equivalent to (36 nmoles) or twice the amount (72 nmoles) of DMBA or DBA substrate. c Specific activity is expressed as pmoles bound/p~ of DN.A/mg of protein/15 rain of incubation at 37°C in the dark. TABLE IV THE E F F E C T OF TOPICAL APPLICATION OF 100 btg OF 7,8-BF AND 2.56 pg OF DMBA ON THE IN VITRO COVALENT BINDING OF T R I T I A T E D DMBA TO DNA BY EPIDERMAL HOMOGENATES FROM THE P R E T R E A T E D MICE a Time after pretreatment
Pretreatment
Spec. act. (× 104) b
Spec. act. (% of controls) c
DMBA DMBA + 7,8-BF DMBA DMBA+ 7,8-BF DMBA DMBA+ 7,8-BF DMBA DMBA+ 7,8-BF DMBA DMBA+ 7,8-BF
4.48 d 5.10 6.21 3.54 8.29 5.14 3.91 4.03 4.11 6.86
100 114 100 57 100 62 100 103 100 167
(h) 1 1 3 3 6 6 12 12 24 24
a The mice were treated topically with 100/~g of 7,8-BF 5 rain before unlabeled DMBA (2.55 #g) and sacrificed at the times indicated. See Materials and Methods for details concerning assay. b Specific activity (Spec. act.) ix expressed as pmoles bound//~g DNA/mg of protein/15 rain of incugation at 37°C in the dark. c Specific activity (Spec. act.) is expressed as percentage of THE DMBA control. d Each value represents 2 experiments with duplicate determinations per experiment.
306 0 ¢i,
0
L
o ~
0
rj
U v
i
~'~
~o
Z o N
~-/
~% %, t'-
"r! '
,~,~
.~
307 Table III notes the effects of adding several AHH modifying agents on the in vitro covalent binding of tritiated DMBA and DBA to DNA b y epidermal homogenates. When added directly to the assay, 7,8-BF and 5,6-BF inhibited the epidermally-mediated covalent binding of [aH] DMBA and [3H] DBA to DNA. Although n o t shown in Table III, 7,8-BF and 5,6-BF had a similar inhibitory effect on the epidermally-mediated convalent binding of [all] MC and [3H] BP to DNA when added directly to the assay tubes. Estradiol and testosterone also inhibited the in vitro binding b u t to a lesser degree than the benzoflavones. Phenobarbital had no effect on AHH activity (Table II) and also had no effect on DMBA and DBA covalent binding to DNA b y epidermal homogenates. Similar binding results were obtained using epidermal microsomes but, because of the large number of mice needed per assay, we decided to use the epidermal homogenates. The in vitro covalent binding of tritiated DMBA to DNA b y epidermal homogenate from 7,8-BF and/or DMBA pretreated mice is shown in Table IV. The specific activity of binding of [3H]DMBA to DNA b y epidermal homogenates in vitro, as a function of time after induction with 2.56/~g of unlabeled DMBA in vivo revealed that the specific activity of binding reached a peak by 6 hours. As can be seen, 7,8-BF was effective in decreasing this in vitro binding only at 3 and 6 hours after pretreatment to the mice. The epidermal homogenates from mice pretreated with 7,8-BF and DMBA 24 h before sacrificing brought a b o u t a high level of in vitro binding than
TABLE
V
T H E E F F E C T O F T O P I C A L A P P L I C A T I O N O F 100 pg O F 7,8-BF A N D 13.4 pg O F M C O N T H E IN V I T R O C O V A L E N T BINDING OF [3H]MC TO DNA BY EPIDERMAL HOMOGENATES FROM THE PRETREATED MICE a Time after p r e treatment (h)
Pretreatment
Spec. act. (× 104 )b
Spec. act. (% of controls) c
1 1 3
MC MC + 7,8-BF MC
1.45 d 1.12 1.62
I00 77 100
3 6
MC + 7,8-BF MC M C + 7,8-BF MC
1.23 4.44 2.95
76 100 66
5.85 5.56 3.74 3.98
1O0 95 100 106
6
12 12 24 24
MC + 7,8-BF MC MC + 7,8-BF
a The mice were treated topically with 100 pg of 7,8-BF 5 rain before unlabeled M C (13.4 pg) and sacrificed at the times indicated. See Materials and Methods for details concerning the in vitro assay. b Specific activity (Spec. act.) is expressed as pmoles bound per pg D N A per m g of protein per 15 rain of incubation at 37°C in the dark. c Specific activity (Spec. act.) is expressed as percentage of the M C control. d Each value represents 2 experiments with duplicate determinations per experiment.
308 TABLE VI T H E E F F E C T O F T O P I C A L A P P L I C A T I O N O F 1 0 0 ~g O F 7 , 8 - B F A N D 56 gg D B A ON T H E IN V I T R O C O V A L E N T B I N D I N G O F [ 3 H ] D B A T O D N A BY E P I D E R M A L H O M O G E N A T E S F R O M T H E P R E T R E A T E D MICE a T i m e a f t e r pret r e a t m e n t (h) 1 1 3 3 6 6
12 12 24 24
Pretreatment
Spec. act. (x 104 ) b
Spec. act. (% of c o n t r o l s ) c
DBA D B A + 7,8-BF DBA D B A + 7,8-BF DBA DBA + 7,8-BF DBA D B A + 7,8-BF DBA D B A + 7,8-BF
1.12 d 1.20 1.23 1.28 2.41 2.46 2.62 2.94 2.10 2.85
100 107 100 104 100 102 100 112 100 136
a T h e m i c e were t r e a t e d t o p i c a l l y w i t h 1 0 0 /~g o f 7 , 8 - B F 5 rain b e f o r e u n l a b e l e d D B A (56 pg) a n d sacrificed a t t h e t i m e s i n d i c a t e d . See Materials a n d M e t h o d s for details conc e r n i n g t h e in v i t r o assay. b Specific a c t i v i t y (Spec. a c t . ) is e x p r e s s e d as p m o l e s b o u n d p e r pg D N A p e r m g o f prot e i n p e r 15 m i n o f i n c u b a t i o n at 3 7 ° C in t h e d a r k . c Specific a c t i v i t y (Spec. act. ) is e x p r e s s e d as p e r c e n t a g e o f t h e D B A c o n t o l . d E a c h value r e p r e s e n t s 2 e x p e r i m e n t s w i t h d u p l i c a t e d e t e r m i n a t i o n s p e r e x p e r i m e n t .
only DMBA pretreatment. In experiments similar to those depicted in Table IV, the in vitro covalent binding of [3H] MC to DNA by epidermal homogenates was also inhibited by early (1--6 h) pretreatment with 7,8-BF and MC as compared to only MC (Table V). However, the in vitro covalent binding of [3HI DBA to DNA by epidermal homogenates was either not significantly changed or was slightly increased by 7,8-BF and DBA pretreatment as compared to DBA only (Table VI). DISCUSSION
Several chemical carcinogens have been shown to bind covalently to cellular nucleic acids and proteins [ 3,6,25--27], and it seems likely that the interaction with one or more of these macromolecules is essential for the carcinogenic process. Previous studies from this laboratory have demonstrated that a correlation exists between tumor initiating ability of several polycyclic hydrocarbons and their ability to bind covalently to DNA in vitro upon incubation with epidermal homogenates and NADPH as the electrophile-generating system [25]. This is in agreement with early reports which showed that a correlation was found between the extent of in vivo binding of several polycyclic hydrocarbons to mouse skin DNA [17] and protein [16] and the carcinogenic activity of the hydrocarbons. In fact, the Millers [3,28] have proposed a significant general theory to explain chemical carcinogenesis.
309 Their theory states that all chemical carcinogens that are not electrophilic reactants must be converted metabolically into a chemically reactive electrophilic form. From the above one would conclude that if the convalent binding of carcinogenic polycyclic hydrocarbons to some critical nucleophile were decreased its carcinogenic activity also would be decreased. In general, this seems to be an accurate deduction, although there are a few situations where the phenomenon becomes unclear. For example, when 7,8-BF and 5,6-BF are added directly to the incubation tubes, they inhibit the epidermally mediated covalent binding of [3HI DMBA and [3H] DBA to DNA. Similar results recently were noted in this laboratory when [3H]BP and [3H]MC were used [29]. Furthermore, Selkirk et al. [30] demonstrated 7,8-BF inhibited the formation of hydroxylated products from BP by rat liver microsome preparations. In addition, experiments in our laboratory using HPLC indicated that when both 7,8-BF and 5,6-BF were added in vitro they inhibited the formation of all BP and DMBA metabolites by rat liver and mouse epidermal microsomes [29,31]. However, as stated earlier our results plus those of Bowden et al. [20] and Kinoshita and Gelboin [9] showed that 7,8BF inhibited the tumor initiating ability of DMBA and MC but either had no effect or enhanced the initiating ability of BP and DBA. 5,6-BF, in general, exhibited a slight inhibitory effect on the tumor initiation of all the hydrocarbons tested [9,18,20]. As shown in Tables IV, V and VI, a correlation was observed between the effects of 7,8-BF on various PAH initiated tumors and the in vitro covalent binding of these PAH to DNA. This relationship was noted when the binding assays consisted of epidermal homogenates from mice treated in the same manner as those used in the tumor initiation experiments. Epidermal homogenates from mice pretreated with both 7,8-BF and either DMBA or MC (1--6 h before sacrificing) had a lesser capability of converting either [3HI MC or [3H] DMBA to a DNA binding product than mice treated with only DMBA or MC. Whereas, the in vitro binding of DBA to DNA was either unchanged or slightly increases when using epidermal homogenates from mice treated with 7,8-BF and DBA as compared to only DBA treated mice. This was true at all time points investigated (Table VI). Recent results reported by Sloane et al. [32--34] may help clarify the somewhat contradictory results reported on the effects of 7,8-BF on PAH carcinogenesis. They demonstrated that model aromatic compounds, including BP, could be metabolized to the aryl hydroxymethyl derivative by either direct hydroxymethylation (independent of cytochrome P-450 dependent mixed function oxidation) or direct oxidation of the aryl side-chain methyl. Also, Sloane [34] found that 7,8-BF activates the aryl hydroxymethyl synthetase of rat liver and lung which catalyzed the hydroxymethylation of BP. Conversely, 5,6-BF had no effect on the synthesis enzyme system [26]. Flesher and Sydnor [35] and Dewhurst et al. [36] reported that 6-hydroxymethyl-BP was a carcinogen. Its tumor initiating ability in mouse skin is currently being investigated.
310 A p r o p o s e d m e t a b o l i c s c h e m e f o r the cellular m e t a b o l i s m o f P A H w h i c h c o u l d a c c o u n t f o r t h e effects o f 7,8-BF and 5,6-BF o n P A H carcinogenesis in m o u s e skin is s h o w n in Fig. 4. A c t i v a t i o n o f P A H to p r o x i m a t e a n d / o r u l t i m a t e c a r c i n o g e n s is r e p r e s e n t e d b y 3 p a t h w a y s . T w o p a t h w a y s , B and C involve o x i d a t i o n and t h e third, A, does n o t involve o x i d a t i o n o f the P A H b y c y t o c h r o m e P-450 d e p e n d e n t o x y g e n a s e s . I f o n e p o s t u l a t e s t h a t the m e t h y l a t e d P A H , D M B A a n d MC, are n o t e f f e c t i v e l y m e t a b o l i z e d via pathw a y A ( h y d r o x y m e t h y l a t i o n ) because o f the presence o f the m e t h y l g r o u p , t h e n a m e c h a n i s m can be p r o p o s e d f o r the m o d i f i c a t i o n o f skin t u m o r i g e n e sis b y 7,8-BF and 5,6-BF. 7,8-BF h a d e i t h e r n o e f f e c t or e n h a n c e s BP a n d D B A skin t u m o r initiation b y partially inhibiting t h e i r o x i d a t i o n b y p a t h w a y C and activating p a t h w a y A. As stated earlier [ 3 4 ] 5,6-BF has n o e f f e c t o n p a t h w a y A ( h y d r o x y m e t h y l a t i o n ) . 7,8-BF drastically c o u n t e r a c t s D M B A and MC skin t u m o r initiation b y p r e v e n t i n g their o x i d a t i o n t o either an e p o x i d e a n d / o r activated m e t h y l g r o u p . 5,6-BF m o d e r a t e l y inhibits D M B A , MC, D B A and BP skin t u m o r initiation b y a similar m e c h a n i s m b u t the differences in m a g n i t u d e o f t h a t i n h i b i t i o n m a y be related to t h e fact t h a t 7,8-BF does n o t i n d u c e e p i d e r m a l A H H activity whereas 5,6-BF does. REFERENCES 1 A.H. Conney, Pharmacological implication of microsomal enzyme induction Pharmacol. REv., 19 (1967) 317. 2 H.V. Gelboin, Carcinogens, enzyme induction, and gene activation, Adv. Cancer Res., 10 (1967) 1. 3 E.C. Miller and J.A. Miller, Biochemical mechanisms of chemical carcinogenesis in the molecular biology of cancer, in H. Busch (Ed.), Academic Press, New York, 1974, pp. 377--402. 4 E. Boyland, The biological significance of metabolism of polycyclic compounds, Symp. Biochem. Soc., 5 (1950) 40. 5 P. Sims, The metabolism of benzo(a)pyrene by rat liver homogenates, Biochem. Pharmacol., 16 (1967) 613. 6 C. Heidelberger, Chemical carcinogenesis, Annu. REv. Biochem., 44 (1975) 79. 7 H.V. Gelboin, A microsome-dependent binding of benzo(a)pyrene to DNA, Cancer Res., 29 (1969) 1272. 8 H.V. Gelboin, F.J. Wiebel and L. Diamond, Dimethylbenzanthracene tumorigenesis and aryl hydrocarbon hydroxylase in mouse skin. Inhibition by 7,8-benzoflavone, Science, 170 (1970) 169. 9 H.B. Gelboin and F.J. Wiebel, Studies on the mechanism of aryl hydrocarbon hydroxylase induction and its role in cytotoxicity and tumorigenesis, Ann. N.Y. Acad. Sci., 170 (1971) 529. 10 K. Burki, A.G. Liebelt and E. Bresnick, Induction of aryl hydrocarbon hydroxylase in mouse tissues from a high and low cancer strain and their F1 hybrids, J. Natl. Cancer Inst., 50 (1973) 369. 11 S. ThompsOn and T.J. Slaga, Mouse epidermal aryl hydrocarbon hydroxylase, J. Invest. Dermatol., 66 (1976) 108. 12 F.J. Wiebel, B.S. Leutz and H.V. Gelboin, Aryl hydrocarbon (benzo(a)pyrene)hydroxylase: A mixed-function oxygenase in mouse, J. Invest. Dermatol., 64 (1975) 184. 13 R.J. Pohl, R.M. Philpot and J.R. Fouts, Cytochome P-450 content and mixed-func-
311
14 15
16
17 18
19
20
21 22 23
24
25
26
27
28 29
30
31
32
tion oxidase activity in microsomes isolated from mouse skin, Drug Metab. Dispos., 4 (1976) 442. A.P. Poland, E. Glover, J.R. Robinson and D.W. Neberg, Genetic expression of aryl hydrocarbon hydroxylase activity 249 (1974) 5599. D.W. Nebert, L.L. Bausserman and R.'R. Bates, Effect of 17~-estradiol and testosterone on aryl hydrocarbon hydroxylase activity in mouse tissues in vivo and in cell culture, Int. J. Cancer, 6 (1970) 470. L.W. Wattenberg and J.L. Leong, Inhibition of the carcinogenic action of 7,12-dimethylbenz(a)anthracene by ~-naphthoflavone, Proc. Soc. Exp. Biol. IVied., 128 (1968) 940. L.W. Wattenberg and J.L. Leong, Inhibition of the carcinogenic action of benzo(a)pyrene by flavones. Cancer Res., 30 (1970) 1922. N. Kinoshita and H.V. Gelboin, The role of aryl hydrocarbon hydroxylase in 7,12dimethylbenz(a)anthracene skin tumorigenesis: on the mechanism of 7,8-benzoflavone inhibition of tumorigenesis, Cancer Res., 32 (1972) 1329. N. Kinoshita and H.V. Gelboin, Aryl hydrocarbon hydroxylase and polycyclic hydrocarbon tumorigenesis: Effect of the enzyme inhibitor 7,8-benzoflavone in tumorigenesis and macromolecular binding, Proc. Natl. Acad. Sci. U.S., 69 (1972) 824. G.T. Bowden, T,J. Slaga, B.G. Shapas and R.K. Boutwell, The role of aryl hydrocarbon hydroxylase in skin tumor initiation by 7,12-dimethylbenz(a)anthracene and 1,2,5,6-dibenzanthracene using DNA binding and thymidine-3H incorporation into DNA as criteria, Cancer Res., 34 (1974) 2634. P.L. Grover and P. Sims, Enzyme-catalyzed reactions of polycyclic hydrocarbons with DNA and protein in vitro,Biochem. J., 110 (1968) 159. K.A.. Burton, Study of the conditions and mechanism of the diphenylamine reaction for the colorimeter estimation of DNA, Biochem. J., 62 (1965) 315. J. Booth, G.R. Keysell and P. Sims, Effects of oestradiol on the in vitro metabolism of 7,12-dimethylbenz(a)-anthracene and its hydroxymethyl derivatives. Biochem. Pharmacol., 23 (1974) 735. R.R. Bates, Sex hormones and skin tumorigenesis. I. Effect of the estrus cycle and castration on tumorigenesis by 7,12-dimethyl(a)anthracene. J. Natl. Cancer Inst., 41 (1968) 559. S.G. Bury, S. Thompson and T.J. Slaga, The role of epidermal aryl hydrocarbon hydroxylase in the covalent binding of polycyclic hydrocarbons to DNA and its relationship to tumor initiation. Biochem. Biophys. Res. Commun., 70 (1976) 1102. C. Heidelberger and M.G. Moldenhauer, The interaction of carcinogenic hydrocarbons with tissue constituents. IV. A quantitative study of the binding to skin proteins of several C 14-labeled hydrocarbons. Cancer Res., 16 (1956) 442. P. Brookes and P.D. Lawley, Evidence for the binding of polynuclear aromatic hydrocarbons to the nucleic acid of mouse skin: relation between carcinogenic power of hydrocarbons and their b i n d i n g t o deoxyribonucleic acid. Nature, 202 (1964) 781. J.A. Miller, Carcinogenesis by chemicals: An overview. G.H.A. Clowes Memorial Lecture. Cancer Res., 30 (1970) 559. T.J. Slaga, D.L. Berry, M.R. Juchau, S. Thompson, S.G. Buty and A. Viaje, Effects of benzoflavones and trichloropropene oxide on polynuclear aromatic hydrocarbon metabolism and initiation of skin tumors in polynuclear aromatic hydrocarbons, in R.I. Freudenthal and D.W. Jones (Eds.), Raven Press, New York, 1976, pp. 127--137. J.K. Selkirk, R.G. Croy, P.P. Roller and H.V. Gelboin, High-pressure liquid chromatographic analysis of benzo(a)pyrene metabolism and covalent binding and the mechanism of action of 7,8-benzoflavone and 1,2-epoxy-3,3,3-trichloropropane, Cancer Res.., 34 (1974) 3474. J. DiGiovanni, T.J. Slaga, D.L. Berry and M.R. Juchau, Metabolism of 7,12-dimethylbenzanthracene in mouse skin homogenates analyzed with high-pressure liquid chromatography. Drug Metab. Dispos., (1977 ) In press. N.H. SIoane and T.K. Davis, Hydroxymethylation of the benzene ring. Microsomal
312 hydroxymethylation of benzo(a)pyrene, Arch. Biochem. Biophys., 163 (1974) 46. 33 N.H. Sloane and M. Heinemann, Hydroxymethylation of the benzene ring. V. NADPH requirement for aryl hydrooxymethylation by a C-1 donor b o u n d to a macromolecule, Biochim. Biophys. Acta., 201 (1970) 384. 34 N.H. Sloane, ~-Naphthoflavone activation of 6-hydroxymethylbenzo(a)pyrene synthetase, 35 (1975) 3731. 35 J.W. Flesher and K.L. Sydnor, Possible role of 6-hydroxymethylbenzo(a)pyrene as a proximate carcinogen of benzo(a)pyrene and 6-methylbenzo(a)pyrene, Int. J. Cancer, 11 (1973) 433. 36 F. Dewhurst, D.A. Kitchen and G. Calcutt, The carcinogenicity of some6-substituted benzo(a)pyrene derivatives in mice. Br. J. Cancer 26 (1972) 506.