dose-response studies were performed with AFBl (10-400 ppb) and 13C. (0-4,000 ... exposure, the results generated a series of parallel AFB 1 dose-response.
TUMOR DOSE-RESPONSE STUDIES WITH AFLATOXIN Bl AND THE AMBIVALENT MODULATOR INDOLE-3-CARBINOL: INHIBITORY VERSUS PRorl'1OTIONAL POTENCY R.H. Dashwood, A.T. Fong, J.D. Hendricks, and G. S. Bailey Department of Food Science and Technology Oregon State University Corvallis, Oregon 97331 ABSTRACT Indole-3-carbinol (l3C), a natural compound from cruciferous vegetables, inhibits aflatoxin B 1 (AFB 1) carcinogenesis in trout when administered prior to and during carcinogen exposure, but also promotes it in the same species when given after AFBl initiation. To provide quantitative potency information for these opposing activities, detailed tumor dose-response studies were performed with AFBl (10-400 ppb) and 13C (0-4,000 ppm). In a plot of (logit) percent tumor response vs log AFBl exposure, the results generated a series of parallel AFB 1 dose-response curves. Increasing 13C doses displaced these curves, respectively, toward higher and lower AFB 1 doses in the inhibition and promotion studies. Similar potencies were observed over the dose range 0-1,500 ppm 13C; the 50% promotion and inhibition (P so and 150) values were 1,000 vs 1,400 ppm 13C, respectively. Differences in the protocols used in the two studies suggest that the inhibitory activity of 13C is more likely to supersede promotion under human exposure conditions. INTRODUCTION The human diet contains many compounds which inhibit the carcinogenic process (4,6,7). However, the overall protective effect which such compounds may exert, and their potential for deliberate chemoprevention, remains difficult to ascertain because unreasonably high doses often are employed in order to elicit an inhibitory effect. Moreover, in some experimental systems, dietary "anticarcinogens" may exhibit promotional activity. For example, indole-3-carbinol (l3C) is a naturally-occurring compound found in cruciferous vegetables (e.g., cabbage and broccoli) which inhibits aflatoxin Bl (AFB 1 ) carcinogenesis in trout when administered prior to and during carcinogen exposure (3,5), but ,which also promotes it in the same species when given after AFBl initiation (1). Since human
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exposure to such dietary agents may be virtually unavoidable, it is important to provide detailed quantitative potency information for these opposing activities (inhibition vs promotion) in order to assess risk vs benefit. Thus, we have undertaken the following tumor studies: (a)
Inhibitory potency. In vivo dose-response analyses were made in trout pre-exposed to 0, 1,000, 2,000, 3,000, or 4,000 ppm 13C in the diet for 4 wk and subsequently treated for 2 wk with the same level of 13C together with dietary AFB 1 in the dose-range 10-320 ppb ("'10,000 animals, Tab. 1). Details of this study have appeared in a recent paper (3).
(b)
Promotional potency. Trout (4,800 animals, Tab. 1) were pretreated with AFB 1 (12.5-400 ppb, 30 min bath exposure) and subsequently fed 0, 750, 1,500, or 2,000 ppm 13C in the diet for 24 wk.
Tab. 1.
Tumor dose-response studies with aflatoxin Bl and indole-3-carbinol. Reprinted from Ref. 3, with permission.
INHIBITORY POTENCY STUDIES PrePost*Percent treatment treatment Tumor (ppm I3C) (ppb AFB1) response
PROMOTIONAL POTENCY STUDIES PrePost*Percent treatment treatment Tumor (ppb AFB1) (ppm I3C) response
0 0 0 0
10 20 40 80
10.7(3.7) 32.2(3.3) 55.6(4.2) 68.4(7.5)
50 100 200 400
0 0 0 0
40.4(7.6) 44.7(1.5) 58.7(7.2) 77.1(2.2)
1000 1000 1000 1000
10 20 40 80
14.7(6.1) 21.1(4.5) 35.2(6.5) 57.6(6.7)
50 100 200 400
750 750 750 750
45.1(5.7) 53.2(1. 9) 65.5(3.6) 84.2(3.5)
2000 2000 2000 2000
20 40 80 160
4.9(1.0) 12.4(1.7) 22.5(7.7) 42.6(7.9)
25 50 100 200
1500 1500 1500 1500
64.7(4.4) 69.9(3.4) 75.8(7.2) 88.6(1.2)
3000 3000 3000 3000
20 40 80 160
1.3(1.2) 2.0(0.8) 3.1(1.9) 5.6(1.2)
12.5 25 50 100
2000 2000 2000 2000
55.2(5.4) 66.7(9.1) 67.9(2.6) 71.5(7.8)
4000 4000 4000 4000
40 80 160 320
0.7(0.5) 1.0(0.8) 5.9(2.0) 7.0(2.3)
*Tumor data are means(+/- SD) from triplicate groups of -100 animals at each AFB1-I3C level. Plotted as logit percent tumor response vs. log AFB1 concentration, a series of parallel curves was produced in both the inhibition and promotion study. I3C-mediated shifts in these curves were calculated in terms of 'percent inhibition' and 'percent promotion', as shown in Figs. 1a and 1b, respectively.
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RESULTS Inhibitory Potency Tumor dose-response data from the inhibition studies are presented in Tab. 1. Plotted as percent tumor response (logit scale) vs log ppb AFB 1 in diet, a series of 5 parallel straight-line curves was evident (see Fig. 2 in Ref. 3). Each increase in 13C dose further offset the position of the logit curve toward higher AFBI dose. From these results, the percent inhibition of AFB 1 tumorigenicity was calculated and plotted as a function of 13C dose (Fig. 1a). Figure 1a serves to highlight the fact that inhibition occurred in a dose-dependent manner, and while complete inhibition was not achieved over the 13C dose-range tested, it was suppressed by 95% at the top dose of 4,000 ppm. Interpolating from Fig. la, the dose producing 50% inhibition of AFBI tumorigenicity (Iso) was 1,400 ppm 13C. PERCENT.%
b) PROMOTION
a) INHIBITION
100
50
2000
4000
750
1500 2000
BC CONCENTRATION (ppm)
Fig. 1.
Percent (a) inhibition and (b) promotion of AFBI tumor incidence versus 13C concentration in diet. Data points represent mean ± S. D. and were calculated from tumor dose-response curves using the formulae: (1 - TDxo/TDxi ) Percent promotion = 100 (1 - TDxi/TDxo)
Percent inhibition = 100
where "x" is the tumor incidence being compared (e.g., 50%), TDxo is the dose of AFBI needed to give that tumor incidence in the 0 ppm (control) group, and TDx. is the dose of AFBI required to produce that tumor incidence \n the group receiving "i" ppm 13C. Reprinted from Ref. 3, with permission.
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Promotional Potency Data from the promotion studies are presented in Tab. 1. When plotted in an analogous fashion to the inhibition data (logit tumor response vs log AFB 1 dose), a series of parallel curves also was produced (not shown). The results for groups receiving 13C were displaced horizontally toward lower AFBl dose. As a result of toxicity observed at the highest 13C dose, 2,000 ppm 13C failed to elicit the greatest promotional response in these studies (Fig. 1b). From Fig. 1b, the concentration of 13C producing 50% promotion (the P so value) was 'V1, 000 ppm 13C, ana the maximum promotional response was 'V75% at the 13C dose of 1,500 ppm. DISCUSSION Inhibitory Versus Promotional Potency The data presented in Fig. 1 indicate that, using the experimental protocols described in these studies, the inhibitory and promotional potencies of 13C were similar at doses 1,500 ppm, some discrepancy was observed due to toxicity (Fig. 1b), and this probably reflects the fact that in the inhibition studies trout received a total of 6 wk exposure to 13C compared with 24 wk postinitiation treatment in the promotion study. These observations highlight an important point, namely, that the inhibitory and promotional potencies of 13C are highly dependent upon the exposure protocols employed. In this context the data in Fig. 1 may be considered misleading, since they imply approximate equivalence of the promotion and inhibition activities--that is, no overall benefit from human exposure to 13C in terms of chemoprevention. The promotion studies reported here, by design, were aimed at evaluating a possible "worst-case scenario" for 13C, i.e., continuous postinitiation exposure to relatively high dietary concentrations. To provide data on the effects of various postinitiation treatments, including those which attempt to mimic likely human exposures, detailed tumor studies have been established in trout pretreated with AFBl and subsequently given 13C either immediately or at various times after terminating AFBl exposure (1, 3, 6, 9 mo) and fed either continuously or for various lengths of time (3, 6, 9 mo, or alternating months or weeks, or Mondays and Thursdays of each week only). Preliminary results indicate that continuous 13C exposure is not a prerequisite for enhancement, although exposure is required over an extended period of time (e.g., 2 days a week for several months; ms. in prep.). Since one week of 13C pretreatment is sufficient for anticarcinogenic activity in trout (unpubl. observ. ), these data collectively suggest that the inhibitory activity of 13C probably supersedes promotion under human exposure conditions. Low-Dose Thresholds Previous studies of 13C inhibition toward AFB1-DNA binding (2,3) produced a potency curve that was linear through the origin at 13C doses
