Time-Dependent Inhibition by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin of Skin Tumorigenesis with Polycyclic Hydrocarbons1

Home , Methylcholanthrene

[CANCER RESEARCH 40. 1580-1587, May 1980] 0008-5472/80/0040-OOOOS02.00 Time-dependent Inhibition by 2,3,7,8-Tetrachlorodibenzo-p-dioxin of Skin Tumorigenesis with Polycyclic Hydrocarbons1

J. DiGiovanni,2 D. L. Berry, G. L. Gleason, G. S. Kishore, and T. J. Slaga

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706 Â¡J. D.. G. S K.J. and Biology Division, Oak Ridge National Laboratory. Oak Ridge. Tennessee 37830 Â¡D.L B . G L G.. T. J. S.J

ABSTRACT reactive intermediates that are capable of interacting covalently with cellular macromolecules (20). The interaction with DNA, a The effects of treating female mice with single topical doses target for activated PAH intermediates, has received consid of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at various times erable attention in recent years and is presently considered a before and after 7,12-dimethylbenz(a)anthracene (DMBA), critical event in the initiation of chemical carcinogenesip. Cur benzo(a)pyrene, 3-methylcholanthrene (MCA), and (Â±)-trans- rent evidence suggests that bay-region diol-epoxides may be 7/S,8Â«-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)- ultimate carcinogenic forms for some PAH (24). This is sup pyrene were studied. TCDD, at doses of 1 /ig/mouse, was ported by recent data concerning BaP and DMBA epoxides applied topically 3 days prior to, 5 min prior to, or 1 day after that produce a DMA-binding product similar to that isolated in the initiator. The application of TCDD 5 min prior to or 1 day vivo after treatment with the parent hydrocarbon (2, 11, 17, after initiation with DMBA, benzo(a)pyrene, or MCA had little 22, 33, 42, 48, 49). In mouse skin, the 7,8-dihydrodiol of BaP or no effect on papilloma formation. In contrast, when TCDD is the only metabolite found to be equally potent or more potent was applied 3 days prior to initiation with these hydrocarbons, as a tumor initiator than BaP itself (30, 46). The potent carci a marked reduction in papilloma formation was observed. nogenic activify BaP-diol-epoxide in newborn mice and mouse TCDD, applied 3 days prior to, 5 min prior to, or 1 day skin lends further support to this concept (26, 44). Additional after initiation with (Â±)-frans-7/?,8Â«-dihydroxy-9a,10a-epoxy- evidence has accumulated in support of "bay-region" diol- 7,8,9,10-tetrahydrobenzo(a)pyrene produced 81, 50, and epoxides as potential ultimate carcinogenic forms for 39% inhibition of papilloma formation after 20 weeks of pro benz(a)anthracene, 7-methylbenz(a)anthracene, chrysene, motion. Similar effects were observed when higher initiating and dibenz(a,/i)anthracene (8, 45, 51-55). doses of DMBA and the diol-epoxide were utilized. In addition, Many factors are known to affect the metabolism of PAH to single i.p. doses of TCDD (1 /xg/mouse) 3 days prior to initiation electrophilic intermediates such as diol-epoxides and conse with DMBA or MCA were highly effective at inhibiting papilloma quently to affect carcinogenic or mutagenic activity. These formation. Several congeners of TCDD, lacking, possessing factors include route of administration, species, sex, age, similar, or possessing different inducing properties, were tested strain, diet, temperature, time of day, season, and the previous for their effects on tumor initiation by DMBA. 3,4,3',4'-Tetra- or concurrent administration of other drugs or environmental chlorobiphenyl effectively inhibited tumor initiation by DMBA, chemicals. TCDD is a highly toxic and widely studied chemical whereas 2,7-dichlorodibenzo-p-dioxin and 2,4,5,2',4',5'-hex- found as an environmental contaminant (for reviews, see Ref. achlorobiphenyl had little or no effect on the tumor response. 41 and the references therein). This compound is known to Topical application of TCDD at the doses utilized in the tumor markedly influence enzyme pathways responsible for both the experiments markedly induced epidermal aryl hydrocarbon activation and inactivation of PAH. TCDD is an extremely potent hydroxylase (21-fold) 3 days after treatment. In addition, epi inducer of hepatic microsomal monooxygenase activity with dermal uridine diphosphoglucuronyltransferase activities were properties similar to MCA (36). In addition, TCDD is capable of stimulated 2- to 3-fold 3 days following TCDD treatment. TCDD inducing AHH (EC 1.14.14.2) of hepatic and extrahepatic treatment had little or no effect on epidermal epoxide hydrase tissues transplacentally (4, 5), in genetically "nonresponsive" or glutathione-S-transferase activities. The time course for mice (40) and in mouse skin (12, 35). UDPGT (EC 2.4.1.17) induction of epidermal aryl hydrocarbon hydroxylase and uri activities are also increased in hepatic (32, 34) and renal (18) dine diphosphoglucuronyltransferase correlated with the time microsomal preparations, as are GST (EC 2.5.1.18) activities course for the inhibitory effects of TCDD on tumor initiation in hepatic cytosolic preparations (1) from rats pretreated with with DMBA, benzo(a)pyrene, and MCA. TCDD. In a preliminary communication (12), we reported that TCDD, INTRODUCTION at doses nontoxic for female CD-1 mice, possessed remarkable inhibitory actions on the initiation of skin papillomas by DMBA Carcinogenic PAH3 require metabolic activation to highly and BaP. The inhibitory effect was dependent on both dose and the time of pretreatment. The present investigation was

Received June 13, 1979; accepted February 8, 1980. ' Research supported by NIH Grants CA-20076, CA-22484, and CA-09020 and by the Department of Energy, under Contract W-7405-eng-26 with the Union TCDD. 2.3.7.8-tetrachlorodibenzo-p-dioxin; MCA. 3-methylcholanthrene; AHH, Carbide Corporation. aryl hydrocarbon hydroxylase; UDPGT, uridine diphosphoglucuronyltransferase; 2 To whom requests for reprints should be addressed. GST, glutathione S-transferase; p-NP. p-nitrophenol; TPA, 12-O-tetradecanoyl- 3 The abbreviations used are: PAH, polycyclic aromatic hydrocarbons; BaP. phorbol-13-acetate; DCDD. 2.7-dichlorodibenzo-p-dioxin; TCB, 3,4,3'.4'-te- benzo(a)pyrene; DMBA, 7.12-dimethylbenz(a)anthracene; BaP-diol-epoxide, trachlorobiphenyl; HCB, 2.4,5,2'.4',5'-hexachlorobiphenyl; EH, epoxide hy (Â±)-rrans-7/i.8a-dihydroxy-9Â«.10<Ã¯-epoxy-7,8.9,10-tetrahydrobenzo(a)pyrene; drase

1580 CANCER RESEARCH VOL. 40

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. Anticarcinogenic Effects of TCDD designed to extend our studies of the anticarcinogenic activity recorded weekly. Papillomas and carcinomas were removed at of TCDD. We have analyzed the effect of treating female mice random for histological verification. The tumor response is with single doses of TCDD at various times before and after presented as the average number of papillomas per mouse. application of 4 tumor initiators. DMBA, BaP, and MCA were Enzyme Assays. Epidermal homogenates were prepared as utilized as PAH carcinogens requiring metabolic activation. described previously (7, 14). Microsomal fractions were ob BaP-diol-epoxide, a proposed ultimate carcinogenic form of tained by centrifuging the whole homogenate at 9,000 x g for BaP, also was utilized as an initiator. In addition, several 20 min and then centrifuging the supernatant at 105,000 x g chlorinated hydrocarbon derivatives related to TCDD, lacking, for 1 hr. Microsomal pellets were suspended once in 0.05 M possessing similar, or possessing different enzyme-inducing Tris-0.25 M sucrose buffer (pH 7.5), resedimented, and then properties, were investigated for their effects on tumor initiation resuspended in the same buffer. by DMBA. Finally, experiments were performed to investigate The AHH assay was a modification of that described by the effects of TCDD on the oxidative and nonoxidative biotrans Juchau ef al. (25). Assays were performed in semidarkness formation pathways for PAH in mouse epidermis. and contained (in a total volume of 1 ml) 0.4 to 1.0 mg microsomal protein, 3 ^mol magnesium chloride, 7.4 /Â¿mol glucose 6-phosphate, 2 units glucose-6-phosphate dehydro MATERIALS AND METHODS genase, 2.4 jumol NADPH, 100 nmol BaP, and 30 /imol potas Chemicals. DMBA, BaP, MCA, NADPH, p-NP, glucose 6- sium phosphate (pH 7.4). Incubations were carried out for 15 phosphate, glucose-6-phosphate dehydrogenase, and uridine min at 37Â°in an atmosphere of 100% O2. Specific activity is 5'-diphosphoglucuronic acid were purchased from Sigma expressed as pmol 3-hydroxybenzo(a)pyrene formed per mg Chemical Co., St. Louis, Mo. BaP-diol-epoxide and 3-hydrox- microsomal protein per min of incubation. ybenzo(a)pyrene were gifts from the Carcinogenesis Program UDPGT activities were measured using a modification of of the National Cancer Institute. [7-"*C]Styrene oxide (9.2 mCi/ previous methods (21, 50) with p-NP as substrate. Typical mmol; 98% pure by gas-liquid chromatography) was supplied incubation mixtures contained (in a final volume of 0.5 ml) 250 by California Bionuclear Corp., Sun Valley, Calif. TCDD (Lot nmol p-NP, 1 jumol uridine 5'-diphosphoglucuronic acid, 37.5 851-144-2; 98.6% pure by gas-liquid chromatography) was jumol Tris-HCI (pH 7.5), and 0.2 to 0.8 mg microsomal protein. supplied by Dow Chemical Co., Midland, Mich. TPA was pur Incubations were carried for 2 hr at 37Â°, and reactions were chased from Dr. Peter Borchert, University of Minnesota, Min terminated by adding 2.5 ml of 0.5 mw trichloroacetic acid. neapolis, Minn. DCDD, TCB, and HCB were purchased from The disappearance of color was monitored at 400 nm as Analabs, Inc., North Haven, Conn. StyrÃ¨ne oxide was obtained described previously (21). Specific activity is expressed as from the Aldrich Chemical Co., Milwaukee, Wis. nmol p-nitrophenyl glucuronide formed per mg microsomal Animals. Female CD-1 mice, purchased from Charles River protein per min of incubation. Breeding Laboratories, North Wilmington, Mass., or female Measurements of microsomal EH (EC 4.2.1.63) and soluble Sencar mice (originally obtained from Dr. R. K. Boutwell, GST activities were performed using a slight modification of the McArdle Laboratory for Cancer Research, University of Wis procedures described by James ef al. (23) with [7-14C]styrene consin, Madison, Wis., and presently raised at Oak Ridge, oxide as substrate. Specific activity for EH is expressed as Tenn.) were utilized for tumor experiments as noted. At 7 to 9 nmol [7-14C]styrene glycol formed per mg microsomal protein weeks of age, the backs of the mice were carefully shaved with per min of incubation, and GST activities are expressed as surgical clippers. Mice were allowed to rest for 2 days, and nmol S-(2-hydroxy-1-phenylethyl)glutathione formed per mg only those mice in the resting phase of the hair cycle were cytosol protein per min of incubation. used. For topical applications, chemicals were applied to the Protein was determined by the method of Lowry ef al. (31) shaved area in 0.2 ml of acetone, and control animals were using bovine serum albumin as standard. Linearity with respect treated with an equal volume of acetone. For some experi to protein concentration and time and saturating concentra ments, TCDD was administered as a single i.p. injection (0.1 or tions of substrate were established using epidermal microsomal 1 /ig/mouse). Special precautions were observed for the han or cytosolic fractions for the assays as described. dling of TCDD and TCDD-pretreated animals. The solutions of TCDD were prepared by introducing sufficient acetone in RESULTS TCDD-containing sealed glass ampuls to make saturated so lutions (0.09 /Â¿gTCDD per ml acetone). The solutions were Effects of Topically Applied TCDD on Tumor Initiation by mixed with corn oil (for i.p. injections) or acetone (for topical DMBA, BaP, MCA, and BaP-diol-epoxide. The effects of single applications) in appropriate concentrations, such that less than topical applications of TCDD at various times relative to initia 0.5 or exactly 0.2 ml, respectively, were administered to each tion with DMBA, BaP, and MCA are shown in Table 1. TCDD mouse. Animals treated with TCDD were kept in rooms separate was applied 3 days prior to, 5 min prior to, or 1 day after the from the control animals to prevent possible contamination of initiator in female Sencar mice. The dose utilized (1 jug/mouse) the controls. produced no visible toxic manifestations. All animals appeared Tumor Experiments. Each experimental group contained 30 normal in terms of weight gain and morphological and histolog preshaved mice. Mice were initiated with either DMBA (10 ical characteristics of the skin after 20 weeks of promotion with nmol), BaP (100 nmol), MCA (100 nmol), or BaP-diol-epoxide TPA. The application of TCDD 5 min prior to or 1 day after (200 nmol). One week after initiation, mice received applica initiation with 10 nmol DMBA produced little or no effect on tions of either 3.4 (Sencar) or 17 (CD-1) nmol TPA twice papilloma formation (Table 1, Experiment 1). In contrast, when weekly. Promotion was continued for 18 to 24 weeks, and the TCDD was applied 3 days prior to initiation with DMBA, a incidence of papillomas and carcinomas was observed and marked reduction (98%) in papilloma formation was observed.

MAY 1980 1581

Table 1 I40 Effect of TCDD application 3 days or 5 min prior to or 1 day following tumor initiation with DMBA, BaP, and MCA Thirty female Sencar mice were used per experimental group. Mice were initiated with 10 nmol DMBA, 100 nmol BaP, or 100 nmol MCA. One week after initiation, mice received 3.4 nmol TPA twice weekly for 20 weeks. Control animals receiving the acetone vehicle followed by TPA promotion had 0.1 papilloma/ mouse. of surviving mice at of con mouse88.70.207.57.28.09.03.60.53.43.73.13.94.21.84.14.5%trol"1002.310096100113100141001081001251004398107 TreatmentExperimenttime-3 20wk30293030293029293029302830293030Papillo-mas/ 1AcetoneTCDD(1)CAcetoneTCDDd)AcetoneTCDDd)Experiment days-3 daysâ€” 5minâ€” 5min+ 1day"+ 1day-3

2AcetoneTCDDd)AcetoneTCDDd)AcetoneTCDDd)Experiment days-3 daysâ€” 5minâ€” 5min+ 1day"+ 1day-5

3AcetoneTCDDd)TCDDd)TCDDd)Treatment min-3 daysâ€” 5min+ 1 day"InitiatorDMBADMBADMBADMBADMBADMBABaPBaPBaPBaPBaPBaPMCAMCAMCAMCANo.-8 -6 -4 -2 a Maximum S.D., 16%. TIME OF APPLICATION IN DAYS (RELATIVE TO INITIATOR) The average number of papillomas per mouse expressed as a percentage of the acetone-pretreated control for each time point. Chart 1. Summary of the time-dependent inhibitory effects of TCDD on tumor c Numbers in parentheses, /jg/mouse. initiation by DMBA (A), BaP (â€¢),and MCA (â€¢).Animals were initiated with 10 TCDD was applied 24 hr after initiator. nmol DMBA, 100 nmol BaP, or 100 nmol MCA and promoted one week later with twice weekly applications of TPA for 20 weeks. TCDD was applied topically (1 jig/mouse) at various times relative to the initiator (abscissa)', tumor response (ordinate) represents the average number of papillomas per mouse expressed as The results, with BaP (100 nmol) and MCA (100 nmol) as the a percentage of the acetone-treated control group for each initiator. 30 mice initiators, were nearly identical (Table 1, Experiments 2 and 3) were utilized for each experimental group. Female CD-1 mice were used for to those obtained with DMBA. TCDD applied 3 days prior to experiments with DMBA and BaP as the initiators, except for experiments when TCDD was given 1 hr after the initiator where female Sencar mice were used. initiation with BaP or MCA reduced the number of papillomas Female Sencar mice were utilized for experiments where MCA was utilized as per mouse by 86 and 57%, respectively. TCDD applied 5 min the initiator. prior to or 1 day after initiation had little or no effect on the average number of papillomas per mouse after 20 weeks of initiating dose of DMBA (10 nmol) utilized in all previous ex promotion. Chart 1 summarizes tumor experiments we have periments. When TCDD was applied 3 or 10 days prior to 100- performed analyzing the time-dependent inhibitory effects of nmol initiating doses of DMBA, a marked inhibition of papilloma TCDD on tumor initiation by these PAH using female CD-1 mice formation was observed (89 and 57%, respectively) after 20 for BaP and DMBA initiation and female Sencar mice for MCA weeks of promotion. The application of TCDD 3 days prior to initiation. Single topical doses of TCDD given as much as 10 initiation with BaP-diol-epoxide (400 nmol) produced a 93% days prior to initiation with DMBA in CD-1 mice still had a inhibition in papilloma formation. marked inhibitory effect. Effects of i.p. Doses of TCDD on Tumor Initiation by DMBA Table 2 illustrates the effects of TCDD treatment on tumor and MCA. Table 4 illustrates the effects of single i.p. doses of initiation with BaP-diol-epoxide in female Sencar mice. The TCDD on tumor initiation by DMBA and MCA in female Sencar initiating dose of BaP-diol-epoxide chosen (200 nmol) was mice. In Experiment 1, animals were initiated with 100 nmol based on previous investigations of the tumor-initiating activity MCA. TCDD (1 jug/mouse) was injected i.p. either 3 days or 5 of this compound in female CD-1 mice (44). As shown Table 2, min before initiation. Control animals received an equal volume TCDD applied 3 days prior to, 5 min prior to, or 1 day after of the corn oil vehicle. When TCDD was injected 3 days before BaP-diol-epoxide produced 81, 50, and 39% inhibition of pap MCA application, an approximately 59% inhibition in papilloma illoma formation, respectively, after 20 weeks of promotion with formation was observed. Injection of TCDD 5 min before MCA TPA. The difference between the average number of papillomas had no effect on the tumor response. In the second set of per mouse in control and experimental groups for each treat experiments, TCDD was injected i.p. at 2 different dose levels ment time were statistically significant (p < 0.01). These ex 3 days prior to initiation with DMBA. Doses of 0.1 or 1 fig TCDD periments have been repeated, yielding similar results. i.p. produced 87 and 92% inhibition, respectively, in DMBA- Table 3 illustrates the inhibitory effect of TCDD pretreatment initiated papilloma formation. Included in both experiments was on female CD-1 mice when higher initiating doses of DMBA an acetone (topical) control group to indicate that both types and BaP-diol-epoxide were utilized. In addition, a control ex of control groups had approximately the same number of periment was included for comparison using the standard papillomas per mouse.

1582 CANCER RESEARCH VOL. 40

Table 2 Table 4 Effect of TCDD application 3 days or 5 min prior to or 1 day following tumor Effect of single i.p. doses of TCDD on tumor initiation by MCA ano DMBA initiation with BaP-diol-epoxide Mice in Experiment 1 were initiated with 100 nmol MCA, and mice in Experi Thirty female Sencar mice were used per experimental group. Mice were ment 2 were initiated with 10 nmol DMBA. Control animals receiving the corn oil initiated with 200 nmol BaP-diol-epoxide. One week after initiation, mice received vehicle followed by TPA promotion had 0.08 papilloma/mouse. 3.4 nmol TPA twice weekly for 20 weeks. Control animals receiving the acetone vehicle followed by TPA promotion had 0.1 papilloma/mouse ofsurvivingmice

of at18wk"3029292829292928Papillo-mas/mouse3.43.91.63.66.16.30.50.8%of con surviv TreatmentExperiment trol01001004192100100813 ingmice menttime-3 at20 1Acetone days-3 TreatmentAcetoneTCDD(1)CAcetoneTCDDd)AcetoneTCDDd)Treatwk302929282628Papillo-mas/mouse31.60.3"1.40.7"1.81.1*%ofcontrol"100191005010061(topical)Corn oilTCDD days-3 days-3 (1fig/mouse)TCDD days-5 days-5 /ig/mouse)Experiment(1 min-3 minâ€” 2Acetone 5min+ (topical)Corn days-3 days-3 1day9+ oilTCDD /ig/mouse)TCDD(1 .0 days-3 1 dayInitiatorBaP-diol-epoxideBaP-diol-epoxideBaP-diol-epoxideBaP-diol-epoxideBaP-diol-epoxideBaP-diol-epoxideNo.(0.1 /ig/mouse)Treatmenttime"-3daysNo. 1 Maximum S.D., 16%. Time of application of TCDD prior to initiation with the hydrocarbon. The average number of papillomas per mouse expressed as a percentage of Thirty female Sencar mice were utilized per experimental group; 1 week after the acetone-pretreated control for each time point. initiation, mice received 3.4 nmol TPA twice weekly for 18 weeks. c Number in parentheses, fig/mouse. c The average number of papillomas per mouse expressed as a percentage of Differences in the number of papillomas per mouse between control (acetone- the i.p. corn oil-pretreated control groups. pretreated) and experimental (TCDD-pretreated) groups are statistically signifi cant ( p < 0.01 ) using Student's f test. * TCDD was applied 24 hr after the initiator. 2.3-fold induction was observed and maintained at 5 and 10 days after TCDD. Table 3 The effect of TCDD, administered i.p. to female CD-1 mice Effect of TCDD pretreatment on higher tumor-initiating doses of DMBA and BaP-diol-epoxide on epidermal AHH and UDPGT, also was determined (data not Thirty female CD-1 mice were used per experimental group. Mice were shown). Each mouse in 3 groups of 25 female CD-1 mice was initiated as indicated and. 1 week following initiation, received 17 nmol TPA twice treated as follows: Group 1, 0.2 ml corn oil i.p.; Group 2, 0.1 weekly for 20 weeks. Control animals receiving the acetone vehicle followed by fig TCDD in 0.2 ml corn oil i.p.; and Group 3, 1.0 /tg TCDD in TPA promotion had 0.03 papilloma/mouse. 0.2 ml corn oil i.p. All mice were sacrificed 3 days later, and No. of surviving epidermal AHH and UDPGT were measured. Injection of 0.1 or Pretreat- Pretreat- mice at mas/ 1.0 jug TCDD i.p. per mouse produced 14- and 17-fold induc mouse33.80 wkAcetonement ment time Initiator 20 tions of epidermal AHH, respectively, when compared to epi -3 days DMBA (10)" TCDD(1)C(10)Acetone -3 days DMBA dermal AHH activity from control mice. UDPGT activities also 2929 0.347.25 9100 were stimulated above control levels approximately equally -3 days DMBA (100) (1.6-fold) at either dose of TCDD. TCDDd)(100)Acetone -3 days DMBA 2930 0.827.54 11100 Effects of TCDD and Congeners of TCDD on Tumor Initi - 10 days DMBA (1 00) ation by DMBA. Several chlorinated hydrocarbon derivatives TCDDd)(100)Acetone -10days DMBA 2828 3.211.53 43100 related to TCDD were tested for their effects on tumor initiation

â€”¿3days BaP-diol-epoxide by DMBA. The results of these experiments are shown in Table (400) 5. TCB (625 fig/mouse) applied topically 3 days prior to TCDDd)7" -3 days BaP-diol-epoxide 29Papillo 0.10%ofcontrol"100 initiation with DMBA produced an approximately 90% inhibition (400)30 in papilloma formation after 24 weeks of promotion. HCB Maximum S.D., 18%. '' The average number of papillomas per mouse expressed as a percentage of pretreatment (625 jug/mouse) 3 days prior to initiation had little the acetone-pretreated control for each group. or no effect on the formation of papillomas following initiation c Number in parentheses, /^g/mouse. with DMBA. DCDD, applied at a dose of 100 jug 3 days prior to Number in parentheses, nmol/mouse. initiation had little or no effect on the tumor response. Table 5 also shows the effects these compounds had on epidermal Effects of TCDD on Epidermal AHH, EH, UDPGT and GST. AHH and UDPGT activities 3 days after a single topical dose. Chart 2 depicts the effects of a single topical dose of TCDD (1 The doses utilized were identical to the doses used for the Â¿tg/mouse) on epidermal AHH, EH, UDPGT, and GST in CD-1 tumor experiments. As illustrated in Table 5, TCDD and TCB mice. Chart 2, A illustrates the effect of TCDD on epidermal induced AHH activity by 21- and 17-fold, respectively, and AHH activity. AHH activity was increased 12-fold 24 hr after UDPGT activity by 2.3- and 2.05-fold, respectively. On the TCDD application, and 72 hr later a 21-fold stimulation was other hand, HCB and DCDD had little effect on epidermal AHH observed. AHH activity also was elevated at 5 and 10 days or UDPGT activities. after TCDD application. Chart 2, B and C, depicts the effects of TCDD on epidermal EH and GST activities, respectively, using [7-14C]styrene oxide as substrate. Topical treatment with DISCUSSION TCDD did not significantly alter epidermal EH or GST activities The toxicity of TCDD in experimental animals has been well at any of the time points monitored. Chart 2D shows that 24 hr established. Recently, several reports have suggested that after treatment with TCDD an approximately 1.4-fold stimula TCDD is a hepatocarcinogen in rats (27, 47). Kouri ef al. (29) tion of UDPGT was observed and by 72 hr an approximately reported that TCDD did not produce sarcomas at the site of

MAY 1980 1583

Chart 2. Effect of TCDD on mouse epider mal AHH (A). EH (6), GST (C), and UDPGT (D). Twenty-five female CD-1 mice were uti lized for each time point, and each curve rep resents an average of 2 separate experiments run in triplicate. Animals were treated topically with 1 Â¿igTCDD and sacrificed 5 min, 1 day, 3 days, 5 days, and 10 days later. Microsomal and cytosolic fractions were prepared as de scribed in "Materials and Methods." Specific activities are expressed as follows: AHH, pmol 3-hydroxybenzo(a)pyrene per mg protein per min of incubation x 10 2; EH, nmol [7-14C]- styrene glycol per mg protein per min of in cubation; GST, nmol S-(2-hydroxy-1-phen- ylethyOglutathione per mg protein per min of incubation; UDPGT. nmol p-nitrophenyl glucuronide per mg protein per min of incubation. The average control value for the 10-day ex periment is represented by a dashed line.

234567 34567 8 9 IO TIME AFTER TCDD (DAYS) TIME AFTER TCDD CDAYS)

Table 5 pretreatment (1 /ng/mouse) 3 to 5 days prior-to initiation almost Effects of TCDD and congeners of TCDD on the tumor-initiating activity of completely abolished the tumor response. However, when BaP DMBA and epidermal AHH and UDPGT activities Thirty female CD-1 mice were used per experimental group. All mice were and MCA were used as the initiators, the magnitude of the initiated with 10 nmol DMBA. Animals were pretreated with single topical doses inhibitory effect was less. The implications of this observation 3 days prior to initiation as follows: TCDD, 1 /ig/mouse; DCDD, 100 fig/mouse; are unknown at present. TCB, 625 ng/mouse; HCB, 625 fig/mouse. One week after initiation, mice The effect of TCDD on tumor initiation by BaP-diol-epoxide received twice weekly applications of 17 nmol TPA for 24 weeks. Control animals receiving the acetone vehicle followed by TPA promotion had 0.03 papilloma/ (a proposed ultimate carcinogenic form of BaP) was somewhat mouse. surprising. TCDD inhibited the formation of papillomas even initiationCompoundTCDDTCBHCBDCDDPapillomas/Effect on DMBA tumor when given 1 day after initiation with the diol-epoxide. This

of con might suggest an alternate mechanism for the inhibitory effect trol"9.0108891AHH"9547025041UDPGT00.9350.8400.4200.430 mouse0.340.43.363.44% of TCDD on BaP-diol-epoxide (other than for DMBA, BaP, and MCA). The structural similarity between acridines, a class of known intercalating agents, and dioxins suggests the possibil ity that TCDD may intercalate between base pairs in DMA. If this were true, other intercalating agents might produce effects Average number of papillomas per mouse expressed as percentage of the DMBA-initiated, TPA-promoted group (3.8 papillomas/mouse). similar to those observed with TCDD. We are currently testing 0 Specific activities are expressed as pmol 3-hydroxybenzo(a)pyrene formed several known intercalating agents on the tumor-initiating ac per mg protein per min of incubation. Specific activity using epidermal micro- tivity of BaP-diol-epoxide. somes from control mice was 45. 0 Specific activities are expressed as nmol p-nitrophenyl glucuronide formed The tumor experiments described in Tables 1, 2, and 4 per mg microsomal protein per min of incubation. Specific activity using epidermal utilized female Sencar mice, whereas, in our previous experi microsomes from control mice was 0.410. ments (12) and in the data presented in Tables 3 and 5 and Chart 1 of the present study, we utilized female CD-1 mice. s.c. injection in C56BL/6 or DBA/2 mice after 36 weeks. The Sencar mice were originally developed using the selective Experiments from our laboratories have shown that TCDD possessed little or no tumor-initiating (16) or tumor-promoting breeding methods described by Boutwell (6). The original skin tumor-susceptible mice developed by Dr. R. K. Boutwell were (3) properties in the skin of CD-1 mice at doses that produced crossed with Charles River CD-1 mice in 1971, and this CD-1 a marked inhibition of the initiation of skin papilloma formation hybrid is what is referred to as Sencar mice in the present by several PAH (12). We therefore chose TCDD as a tool for study. Female Sencar mice are 20 to 80 times more sensitive studying the effects of enzyme induction on tumor initiation by than female CD-1 mice to skin tumorigenesis by DMBA4 and in PAH in mouse skin. addition are more sensitive to skin tumorigenesis by BaP.4 The present investigation was designed to extend our earlier However, no significant differences were observed with respect findings that TCDD (at nontoxic doses) possessed remarkable to basal or inducible levels of epidermal monooxygenase activ anti-tumor-initiating activity in CD-1 mice. In the present study, ity between Sencar and CD-1 mice.4 Our results presented TCDD was applied topically before and after tumor initiation here indicate that female Sencar mice respond in a manner with 3 PAH carcinogens requiring metabolic activation (DMBA, identical to female CD-1 mice under the influence of TCDD (for BaP, and MCA). The inhibitory effect of TCDD was highly both the inhibitory effect and the time dependence of this dependent on the time of treatment relative to the initiator. effect). Furthermore, the data suggest that the inhibitory effect Maximal inhibition of tumor initiation was observed when TCDD was given 3 to 5 days before the initiator. Little or no effect " J. DiGiovanni, T. J. Slaga, and R. K. Boutwell. Comparison of the tumor- was observed when TCDD was given 5 min before or 1 day initiating activity of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene in fe after initiation. When DMBA was used as the initiator, TCDD male Sencar and CD-1 mice, submitted for publication.

1584 CANCER RESEARCH VOL. 40

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. Anticarcinogenic Effects of TCDD of TCDD may be a generalized phenomenon in mice that are found previously that the quantity of total [3H]DMBA bound to sensitive to skin tumorigenesis by PAH. DMA and RNA of the epidermis was decreased in animals Recent work by Kouri ef al. (28, 29) regarding the effects of treated with TCDD. Furthermore, a recent study by Cohen ef s.c. coinjection of TCDD on MCA-induced fibrosarcomas sug al. (9) indicated that although the total binding of BaP to gested a potential enhancing effect in certain mouse strains; epidermal DMA was increased in mice pretreated with TCDD, hence, we determined the effects of i.p. administered TCDD on the major hydrocarbon-deoxyribonucleoside adduci present in DMBA- and MCA-initiated skin papillomas in female Sencar the DMA of control mice (i.e., BaP-diol-epoxide bound to the mice. The results presented in Table 4 indicated that TCDD, exocyclic amino group of guanine) was absent. These results administered i.p., effectively inhibited tumor initiation with both suggest that significant differences exist in the metabolic acti initiators. In addition, i.p. administered TCDD, even at doses as vation or detoxification of DMBA versus BaP. low as 0.1 /ig/mouse (4 /Â¿g/kg),effectively induced epidermal To further analyze the role of enzyme induction in the anti- AHH and UDPGT activities 3 days after injection. These results carcinogenic effects of TCDD, several closely related conge correlated very well with the tumor data in Table 4. ners were tested for their effects on tumor initiation with DMBA. This unusually potent, time-dependent inhibitory action of TCB and HCB are pure polychlorinated biphenyl isomers pos TCDD on skin tumor initiation by PAH stands in contrast to the sessing, respectively, MCA- and phenobarbital-type inducing resports by Kouri et al. (28, 29). In those studies, TCDD was properties (19, 38). Induction of microsomal monooxygenase found to enhance the formation of MCA-induced fibrosarcomas enzymes with phÃ©nobarbital is characterized by increased con at the site of s.c. injection when the 2 compounds were admin centrations of cytochrome(s) P-450. Furthermore, phÃ©nobar istered concurrently to "nonresponsive" DBA/2 mice. This bital enhances the metabolism of a wide variety of substrates effect was attributed to the ability of TCDD to enhance the (10). On the other hand, microsomal monooxygenase induction metabolism of MCA in s.c. tissues. However, in their study, with MCA is characterized by increased cytochrome(s) Pi-450, when TCDD was administered i.p. at a dose of 100 jig/kg 2 a cytochrome(s) spectrally distinct from P-450, and an altera days prior to s.c. injection of MCA in DBA/2 mice, no effect tion in substrate specificity (10, 43). TCB, but not HCB, had a was observed. These results would tend to suggest that the marked inhibitory effect at a dose that significantly induced enhancing effect of TCDD on MCA-induced fibrosarcomas is epidermal AHH and UDPGT. It is interesting to note that TCB unrelated to induction of AHH, since this dose (which was 25- and TCDD are approximate stereoisomers; however, TCB is fold higher than the 4-jug/kg dose utilized in our experiments) over 400-fold less potent than TCDD for inducing hepatic AHH would be expected to induce skin AHH in DBA/2 mice (37) 2 in chicken embryos and over 4000-fold less potent for inducing days after treatment. Additional work from our laboratories (13) hepatic AHH following i.p. administration to C57BL/6J mice demonstrated that i.p. injection of TCDD in CD-1 mice effec (38). In addition, TCB is a nearly planar molecule, similar to tively induced microsomal monooxygenases in other extrahe- TCDD. As shown in Table 4, TCB (at an approximately 600- patic tissues such as the kidney and the lung. These results as fold higher dose), was quite effective at inhibiting tumor initia well as those presented in this study suggest that when admin tion by DMBA. DCDD lacks the ability to induce AHH (39), and istered i.p., TCDD is rapidly distributed to a variety of tissues, this could explain the inability to inhibit tumor initiation with including skin at levels more than adequate to effect induction DMBA. of AHH and other microsomal enzymes. The results presented above, coupled with the observed The induction of s.c. fibrosarcomas is a measure of the increases in epidermal AHH and UDPGT, suggested a possible complete carcinogenic (i.e., initiating and promoting compo mechanism for the ability of TCDD to inhibit tumor initiation by nents) of the test carcinogen. When modifying factors are PAH. Under the influence of TCDD, epidermal cells may be tested in systems such as these, it is extremely difficult to programmed to inactivate PAH carcinogens more efficiently determine whether the effect resides at the level of initiation or and therefore eliminate them more readily. Further analysis of promotion. However, by using the 2-stage system of skin the profile of water-soluble metabolites as well as of rates of tumorigenesis, one can study the effects of modifying factors clearance and excretion from epidermal cells may shed fur- on the initiation and promotion components separately and therlight on the inhibitory effect of TCDD and similar com independently. The results presented in our study indicate that pounds. the inhibitory action of TCDD on skin tumorigenesis by PAH REFERENCES carcinogens is an effect on the initiation phase. Induction of microsomal enzyme systems in several tissues 1. Baars, A. J., Jansen, M., and Breimer, D. D. The influence of phÃ©nobarbital, 3-methylcholanthrene and 2,3.7,8-tetrachlorodibenzo-p-dioxin on glutathi- has been postulated as a mechanism for the anticarcinogenic one-S-transferase activity of rat liver cytosol. Biochem. Pharmacol., 27. effects of a wide variety of compounds including chlorinated 2487-2494. 1978. 2. Baird. W. M.. and Dipple, A. Photosensitivity of DNA-bound 7,12-dimethyl- hydrocarbons (Ref. 15 and the references therein). However, benz(a)anthracene. Int. J. Cancer. 20 427-431, 1977. these studies have not demonstrated conclusively that the 3. Berry. D. L . DiGiovanni. J.. Juchau. M R.. Bracken, W. M.. Gleason. G. L., inhibitory actions are a result of induction within the target and Slaga. T. J. Lack of tumor-promoting ability of certain environmental chemicals in a two-stage mouse skin tumorigenesis assay. Res. Commun. tissues. During the course of our experiments with TCDD, it Chem. Pathol. Pharmacol., 20 101-108, 1978. was observed that the time course of the inhibitory effect on 4. Berry. D. L., Slaga, T. J., Wilson, N. M.. Zachariah. P. K.. Namkung, M. J.. tumor initiation correlated with the magnitude as well as the Bracken, W M., and Juchau, M. R. Transplacental induction of mixed function oxygenases in extra-hepatic tissues by 2.3.7,8-tetrachlorodibenzo- time course for induction of monooxygenase activity in the skin p-dioxin. Biochem. Pharmacol., 26. 1383-1388. 1977. (35, 40). As illustrated in Charts 1 and 2 of the present study, 5. Berry, D. L., Zachariah, P. K., Namkung, M. J., and Juchau, M. R. Transpla cental induction of carcinogen-hydroxylating systems with 2,3.7,8-tetra- the time course for the inhibitory effects of TCDD on DMBA, chlorodibenzo-p-dioxin. Toxicol. Appi. Pharmacol.. 36. 569-584, 1976. BaP, and MCA tumor initiation correlated with the time course 6. Boutwell, R. K. Some biological aspects of skin carcinogenesis. Prog. Exp. for induction of epidermal AHH and UDPGT. In addition, we Tumor Res.. 4: 207-250, 1964.

MAY 1980 1585

7. Bowden. G. T., Slaga, T. J., Shapas, B. G. and Boutwell, R. K. The role of duced cancers in inbred strains of mice. In: R. Freudenthal and P. Jones aryl hydrocarbon hydroxylase in skin tumor initiation by 7.12-dimethyl- (eds.), Carcinogenesis, A Comprehensive Survey, Vol. 1, pp. 139-151. benz[a)anthracene and 1,2,5,6-dibenzanthracene using DMA binding and 29. Kouri. R. E., Rude, T. H., Joglekar, R.. Dansette, P. M., Jerina, D. M.. Atlas, thymidine-3H incorporation into DMA as criteria. Cancer Res., 34: 2634- S. A., Owens, I. S., and Nebert, D. W. 2,3,7,8-Tetrachlorodibenzo-p-dioxin 2642,1974. as co-carcinogen causing 3-methylcholanthrene-initiated subcutaneous tu mors in mice genetically "nonresponsive" at Ah locus. Cancer Res., 38: 8. Chouroulinkov, I., Gentil, A., Tierney, B., Grover, P., and Sims, P. The metabolic activation of 7-methylbenz(a)anthracene in mouse skin: high tu 2777-2783, 1978. mor-initiating activity of the 3,4-dihydrodiol. Cancer Lett.. 3 247-253, 30. Levin, W., Wood, A. W., Chang, R. L., Slaga, T. J., Yagi, H., Jerina, D. M., 1977. and Conney, A. H. Marked differences in the tumor-initiating activity of 9. Cohen, G. M., Bracken. W. M., Iyer, P. R., Berry, D. L., Selkirk, J. K., and optically pure ( + )- and (â€”Mrans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene Slaga, T. J. Anticarcinogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on mouse skin. Cancer Res., 37: 2721-2725, 1977. on benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene tumor initiation and 31. Lowry, O. H., Rosebrough, N. J., Farr. A. L., and Randall, R. J. Protein its relationship to DMA binding. Cancer Res., 39: 4027-4033, 1979. measurement with the Folin phenol reagent. J. Biol. Chem., 793. 265-275, 10. Conney, A. H. Pharmacological implications of microsomal enzyme induc 1951. tion. Pharmacol Rev., 79; 317-366, 1967 32. Lucier, G. W., McDaniel, O. S., and Hook, G. E. R. Nature of the enhance 11. Daudel, P., Duquesne. M.. Vigny. P.. Grover, P. L.. and Sims, P. Fluores ment of hepatic uridine diphosphate glucuronyltransferase activity by cence spectral evidence that benzo(a)pyrene-DNA products in mouse skin 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Biochem. Pharmacol.. 24:325- arise from diol-epoxides. FEBS Lett., 57. 250-253, 1975. 334, 1975. 12. DiGiovanni. J., Berry, D. L., Juchau. M. R. and Slaga, T J. 2,3,7.8-Tetra- 33. Moschel, R. C., Baird, W. M., and Dipple, A. Metabolic activation of the chlorodibenzo-p-dioxin: potent anticarcinogenic activity in CD-1 mice. Bio- carcinogen 7,12-dimethylbenzo(a)anthracene for DNA binding. Biochem. chem. Biophys. Res. Commun., 86. 577-584, 1978. Biophys. Res. Commun.. 76: 1092-1098, 1977. 13. DiGiovanni, J.. Berry, D. L., Slaga, T. J., Jones. A. H., and Juchau, M. R 34. Owens, I. S. Genetic regulation of UDP-glucuronosyltransferase induction Effects of pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin on the ca by polycyclic aromatic compounds in mice. J. Biol. Chem., 252: 2827- pacity of hepatic and extrahepatic mouse tissues to convert procarcinogens 2833, 1977. to mutagens for Salmonella typhimurium auxotrophs. Toxicol. Appi. Phar 35. Pohl, R. J., Philpot, R. M., and Fouts, J. R. Cytochrome P-450 content and macol., 50. 229-239, 1979. mixed-function oxidase activity in microsomes isolated from mouse skin. 14. DiGiovanni, J., Slaga, T. J.. Berry, D. L., and Juchau, M. R. Metabolism of Drug Metab. Dispos., 4: 442-450, 1976. 7,12-dimethylbenz(a]anthracene in mouse skin homogenates analyzed with 36. Poland, A., and Glover, E. Comparison of 2,3.7.8-tetrachlorodibenzo-p- high pressure liquid chromatography Drug Metab. Dispos., 5: 295-301. dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methyl 1977. cholanthrene. Mol. Pharmacol., 70: 349-359, 1974. 15. DiGiovanni, J., Slaga, T. J.. Berry. D. L.. and Juchau, M. R. Inhibitory effects 37. Poland, A., and Glover, E. Genetic expression of aryl hydrocarbon hydrox of environmental chemicals on polycyclic aromatic hydrocarbon carcinogen- ylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin: evidence for a receptor mu esis. In: T. J. Slaga (ed.), Modifiers of Chemical Carcinogenesis, pp. 145- tation in genetically non-responsive mice. Mol. Pharmacol., 7 7: 389-398, 168. New York: Raven Press, 1980. 1975. 16. DiGiovanni, J., Viaje, A., Berry. D. L., Slaga, T. J., and Juchau, M. R. Tumor- 38. Poland, A., and Glover, E. Chlorinated biphenyl induction of aryl hydrocar initiating ability of 2.3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor bon hydroxylase activity: a study of the structure-activity relationship. Mol. 1254 in the two-stage system of mouse skin carcinogenesis. Bull. Environ. Pharmacol., 73:924-938, 1977. Contam. Toxicol., 18: 552-557, 1977. 39. Poland. A., Glover, E., and Kende, A. Stereospecific, high affinity binding of 17. Dipple, A., and Nebzydoski, J. A. Evidence for the involvement of a diol- 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. J. Biol. Chem., 257: epoxide in the binding of 7,12-dimethylbenz(a)anthracene to DMA in cells in 4936-4946, 1976. culture. Chem.-Biol. Interact.. 20. 17-26. 1978. 40. Poland, A. P., Glover, E., Robinson, J. R., and Nebert, D. W. Genetic 18. Fowler, B. A., Hook, G. E. R., and Lucier. G. W. Tetrachlorodibenzo-p-dioxin expression of aryl hydrocarbon hydroxylase activity: induction of monoox- induction of renal microsomal enzyme systems: ultrastructural effects on ygenase activities and cytochrome P-450 formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice genetically "nonresponsive" to other aromatic pars recta (S3) proximal tubule cells of the rat kidney. J. Pharmacol. Exp. Ther., 203. 712-721. 1977. hydrocarbons. J. Biol. Chem.. 249: 5599-5606, 1974. 19. Goldstein, J. A.. Hickman. P., Bergman, H., McKinney, J. D., and Walker, 41. Poland, A., and Kende, A. 2,3,7,8-Tetrachlorodibenzo-p-dioxin: environ M. P. Separation of pure polychlorinated biphenyl isomers into two types of mental contaminant and molecular probe. Fed. Proc., 35: 2404-2411, inducers on the basis of induction of cytochrome P-450 or P-448. Chem.- 1976. Biol. Interact., 17: 69-87. 1977. 42. Sims, P., Grover, P. L., Swaisland, A., Pal, K., and Hewer, A. Metabolic 20. Heidelberger. C. Chemical carcinogenesis. Annu. Rev. Biochem., 44: 79- activation of benzo(a)pyrene proceeds by a diol-epoxide. Nature (Lond.), 121.1975 252. 326-328. 1974. 21. Hollmann, S., and Touster. O Alterations in tissue levels of uridine diphos- 43. Sladek, N. E., and Mannering, G. J. Evidence for a new P-450 hemoprotein phate glucose dehydrogenase, uridine diphosphate glucuronic acid pyro- in hepatic microsomes from methylcholanthrene treated rats. Biochem. phosphatase and glucuronyltransferase induced by substances influencing Biophys. Res. Commun., 23. 668-674. 1966. the production of ascorbic acid. Biochim. Biophys. Acta, 26. 338-352, 44. Slaga, T. J., Bracken, W. M., Gleason, G., Levin, W., Jerina, D. M., and 1962. Conney, A. H. Marked differences in the skin tumor-initiating activities of the 22. Ivanovic. V., Geacintov, N. E., Jeffrey. A. M., Fu, P. P.. Harvey, R. G., and optical enantiomers of the diastereisomeric benzo(a)pyrene 7,8-diol-9,10- Weinstein, I. B. Cell and microsome mediated binding of 7,12-dimethyl- epoxides. Cancer Res., 39: 67-71, 1979. benz(a)anthracene to DMA studies by fluorescence spectroscopy. Cancer 45. Slaga, T. J., Huberman, E.. Selkirk. J. K., Harvey, R. G., and Bracken, W. Lett., 4: 131-140, 1978. M. Carcinogenicity and mutagenicity of benz(a)anthracene diols and diol- 23. James. M. O., Fouts, J. R.. and Bend, J. R. Hepatic and extrahepatic epoxides. Cancer Res., 38: 1699-1704, 1977. metabolism, in vitro, of an epoxide (8-'"C-styrene oxide) in the rabbit. 46. Slaga, T. J., Viaje, A., Berry, D. L., Bracken, W. M., Buty. S. G.. and Biochem. Pharmacol., 25. 187-190. 1976. Scribner, J. D. Skin tumor-initiating ability of benzo(a)pyrene-4,5,7.8- and 24. Jerina, D. M., Lehr, R., Schaefer-Ridder. M., Yagi. H.. Karle. J. M.. Thakker, 7,8-diol-9,10-epoxidesand 7,8-diol. Cancer Lett., 2. 115-122, 1976. D. R., Wood, A. W., Lu, A Y. H., Ryan, D., West. S.. Levin. W., and Conney, 47. Van Miller, J. P., Lalich, J. J., and Allen, J. R. Increased incidence of A. H. Bay-region epoxides of dihydrodiols: a concept explaining the muta- neoplasms in rats exposed to low levels of 2,3,7,8-tetrachlorodibenzo-p- genie and carcinogenic activity of benzo[a]pyrene and benzo[a]anthracene. dioxin. Chemosphere, 70: 625-632, 1977. In: H. H. Hiatt. J. D. Watson, and J. A. Winsten (eds.), Origins of Human 48. Vigny, P., Duquesne, M., Coulomb, H., Tierney. B.. Grover. P. L., and Sims. Cancer, pp. 639-658. Cold Spring Harbor. N.Y.: Cold Spring Harbor Lab P. Fluorescence spectral studies on the metabolic activation of 3-methyl oratory. 1977. cholanthrene and 7,12-dimethylbenz(a)anthracene in mouse skin. FEBS 25. Juchau, M. R., Pedersen, M. G., and Symms, K. G. Hydroxylation of 3,4- Lett., 82: 278-282, 1977. benzpyrene in human fetal tissue homogenates. Biochem Pharmacol.. 21: 49. Weinstein, I. B.. Jeffrey, A. M.. Jenette. K. W., Blobstein, S. H., Harvey, R. 2269-2272. G., Harris. C.. Autrup. H., Kasai, H.. and Nakanishi, K. Benzo(a)pyrene diol 26. Kapitulnik, J., Levin, W., Conney. A. H.. Yagi, H., and Jerina. D. M. epoxides as intermediates in nucleic acid binding in vitro and in vivo. Science Benzo(a)pyrene-7,8-dihydrodiol is more carcinogenic than benzo(a)pyrene (Wash. D. C.), 793. 592-595. 1976. in newborn mice. Nature (Lond.), 266. 378-380, 1977. 50. Wisnes, A. Studies on the activation in vitro of glucuronyltransferase. 27. Kociba, R. J.. Keyes, D. G., Beyer, J. E., Carreon, R. M., Wade, C. E., Biochim. Biophys. Acta, Ã•97:279-291, 1969. Dittenber, D. A., Kalnins, R. P., Franson, L. E., Park, C. N.. Barnard, S. D., 51. Wood, A. W.. Chang, R. L., Levin, W.. Lehr, R. E., Schaefer-Ridder, M., Hummel, R. A., and Humiston, C. G. Results of a two year chronic toxicity Karle, J. M., Jerina. D. M., and Conney, A. H. Mutagenicity and cytotoxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in of benzo(a)anthracene diol epoxides and tetrahydroepoxides: exceptional rats. Toxicol. Appi. Pharmacol., 46: 279-305, 1978. activity of the bay region 1,2-epoxides. Proc. Nati. Acad. Sci. U.S.A., 74: 28. Kouri, R. E. Relationship between levels of aryl hydrocarbon hydroxylase 2746-2750, 1977. activity and susceptibility to 3-methylcholanthrene and benzo(a)pyrene-in- 52. Wood, A. W., Levin. W., Chang, R. L.. Lehr, R. E., Schaefer-Ridder, M.,

1586 CANCER RESEARCH VOL. 40

Karle, J. M., Jerina, D. M., and Conney, A. H. Tumorigenicity Ã¶l five 54. Wood. A. W.. Levin, W., Ryan. D., Thomas. P. E., Yagi, H., Moh, H. D , dihydrodiols of benz(a)anthracene on mouse skin: exceptional activity of Thakker. D. R., Jerina, D. M.. and Conney, A. H. High mutagenicity of benz(a)anthracene-3.4-diol. Proc. Nati. Acad. Sci. U.S.A., 74: 3176-3179. metabolically activated chrysene-1,2-dihydrodiol: evidence for bay region 1977. activation of chrysene. Biochem. Biophys. Res. Commun., 78: 847-854, 53. Wood, A. W.. Levin. W., Lu, A Y. H.. Ryan. D , West, S. B.. Lehr, R. E., 1977 Schaefer-Ridder, M., Jerina, D. M., and Conney, A. H. Mutagenicity of 55. Wood. A. W.. Levin. W., Thomas. P. E.. Ryan. D., Karle. J. M.. Yagi. H.. metabolically activated benz(a)anthracene-3,4-dihydrodiol: evidence for bay Jerina. D M , and Conney, A. H. Metabolic activation of dibenzo(a.ri)- region activation of carcinogenic polycyclic hydrocarbons. Biochem. Bio- anthracene and its dihydrodiols to bacterial mutagens Cancer Res . 36. phys. Res. Commun.. 72: 680-686. 1976. 1967-1973, 1978.

MAY 1980 1587

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. Time-dependent Inhibition by 2,3,7,8-Tetrachlorodibenzo-p -dioxin of Skin Tumorigenesis with Polycyclic Hydrocarbons

J. DiGiovanni, D. L. Berry, G. L. Gleason, et al.

Cancer Res 1980;40:1580-1587.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/40/5/1580

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/40/5/1580. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.