Fd Chem. Toxic. Vol. 29, No. 6, pp. 391-400, 1991 Printed in Great Britain. All rights reserved

0278-6915/91 $3.00 + 0.00 Copyright © 1991 Pergamon Press pie

CHEMOPROTECTIVE A N D HEPATIC ENZYME INDUCTION PROPERTIES OF INDOLE A N D I N D E N O I N D O L E ANTIOXIDANTS IN RATS H. G. SHERTZER* Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, OH 45267-0056, USA and M. SAINSBURY School of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK

(Accepted 19 February 1991) Abstract--Three indole antioxidants were compared for their efficacy to inhibit lipid peroxidation, prevent chemical hepatotoxicity and induce enzyme systems involved in the biotransformation of xenobiotics. The dietary indolyl compound indole-3-carbinol (I-3-C), and the synthetic compounds 5,10-dihydroindeno[1,2-b ]indole (DHII) and 4b,5,9b, 10-tetrahydroindeno[l,2-b]indole (THII) inhibited carbon tetrachloride (CC14)initiated lipid peroxidation in rat-liver microsomes, with the order of efficacy THII > DHII = butylated hydroxytoluene (BHT)>> I-3-C. Each of the indole compounds protected isolated rat hepatocytes against toxicity by CC14, N-methyl-N'-nitro-N-nitrosoguanidine and methylmethanesulphonate (THII = DHII >>I-3-C). In vivo administration of the indole compounds 1 hr before treatment with CC14 protected against hepatotoxicity (THII > DHII > I-3-C). For the enzyme induction studies, phenobarbital and fl-naphthoflavone were used as standards, with corn-oil vehicle controls. The compounds were administered by gavage at 50 mg/kg body weight/day for 10 days. I-3-C produced increases in levels of hepatic cytochromes P-450 and ethoxyresorufin O-deethylase (EROD) activity, as well as in UDP-glucuronosyl transferase (UDPGT), glutathione S-transferase (GST), glutathione reductase (GSSG-Red) and quinone reductase. I-3-C produced decreased glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities. DHII produced increases in EROD, UDPGT, GST, GSSG-Red and quinone reductase, with decreases in NDMA-demethylase and GSH-Px activities. The only observed effect of THII was a modest induction of EROD activity. After treatment with the indole compounds for 10 days, I-3-C enhanced, while DHII diminished, CC14-mediated 24-hr hepatotoxicity in rats. We conclude that DHII and THII are suitable candidates to develop further as potential chemoprotective and therapeutic agents for use in humans to treat disorders involving free radicals. THII has the greater radical scavenging efficacy, whereas DHII has the greater capacity to induce many different antioxidative enzymes.

INTRODUCTION H u m a n s are constantly exposed to a complex blend of toxicants and carcinogens on the one hand, and chemoprotective agents on the other (Ames, 1983; Wattenberg, 1985). The balance between intoxication and detoxification normally favours the latter because of the existence of several protective mechanisms including the presence of endogenous and xenobiotic scavengers of radicals and electrophiles, and the *To whom all correspondence should be addressed.

Abbreviations: ALK = alkaline phosphatase; ALT = alanine aminotransferase; BHT = butylated hydroxytoluene; DHII = 5,10-dihydroideno[1,2-b]indole; DMSO = dimethylsulphoxide; EROD = ethoxyresorufin O-deethylase; GSH-Px = glutathione peroxidase; GSSG-Red= glutathione reductase; GST = glutathione-S-transferase; I-3-C = indole-3-carbinol; MMS = methyl methanesulphonate; MNNG = N-methyl-N'-nitro-N-nitrosoguanidine; NDMA = N-nitrosodimethylamine; OTC = ornithine transcarbamylase; quinone reductase = NAD(P)H : (quinone acceptor)oxidoreductase; S O D = superoxide dismutase; THII = 4b,5,9b,10-tetrahydroindeno[1,2-b]indole; UDPGT = UDP-glucuronosyl transferase. 391

presence of many detoxifying enzymes. There appears to be a relationship between the ability of certain xenobiotics (such as sets of phenolic antioxidants or azo dyes) to act as anticarcinogens, and their ability to induce a set of enzymes involved in carcinogen detoxification and excretion (Talalay et al., 1988). These include the non-oxidative enzymes UDP-glucuronosyl transferase ( U D P G T ) , glutathione S-transferase (GST), and N A D ( P ) H : (quinone acceptor)oxidoreductase (quinone reductase); these and other enzymes comprise a set, the co-ordinated expression of which is in part under the control of the aryl hydrocarbon hydroxylase [Ah] gene locus. These enzymes appear to be up-regulated in part by the interaction of a trans-acting Ah-receptor-ligand complex with a binding locus in the 5' flanking region of the [Ah] gene locus, and constitutively down-regulated by both cis- and trans-acting factor(s) (Nebert et al., 1990). It is not clear whether other enzymes, particularly those involved in moderating oxidative stress (such as superoxide dismutase and glutathione peroxidase and reductase), are part of this gene battery. Certain indole antioxidants such as indole-3-carbinol (I-3-C),

392

H. G. SHERTZERand M. SAINSBURY

5,10-dihydroindeno[l,2-b]indole (DHII) and 4b,5,9b, 10-tetrahydroindeno[1,2-b]indole (THII) have been shown to be protective against hepatotoxicity in vitro and in vivo (Shertzer and Sainsbury, 1988; Shertzer et al., 1987a,b and 1991). Although DHII and THII are both indenoindoles, they are quite distinct in chemical structure. DHII is planar with the indole and indene moieties conjugated, while THII is non-planar with the indoline and indan moieties unconjugated. In this paper we evaluate the oxidative and nonoxidative enzyme induction capabilities for these indole protectants, as well as their antioxidant efficacies, in relation to their abilities to protect against chemical hepatotoxicity. MATERIALS AND METHODS

Chemicals. Enzymes, substrates, co-factors and other chemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA), except when noted below. Mazola, the corn-oil vehicle, was processed as described previously (Shertzer and Tabor, 1985) to remove tocopherols and antioxidant activity. Methyl methanesulphonate (MMS), I-3-C, CC14, 4-dimethylaminoantipyrine and N-nitrosodimethylamine (NDMA) were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). Collagenase was obtained from Worthington Biochemicals (Freehold, N J, USA). 7-Ethoxyresorufin was obtained from Pierce Chemical Co. (Rockford, IL, USA), while sodium pentobarbital (65 mg/ml) was from W. A. Butler Co. (Columbus, OH, USA), and fl-naphthoflavone was from Eastman Organic Chemicals (Rochester, NY, USA). DHII (Shertzer and Sainsbury, 1988) and THII (Shertzer et al., 1991) were synthesized as described previously. Animals and tissue preparation. Male Wistar rats (200-250 g, Harlan Sprague-Dawley Inc., Indianapolis, IN, USA) were housed individually, using hardwood chip bedding, and maintained on Teklad LM-485 rodent diet (Teklad Mills, Madison, WI, USA). The rats were kept in an environmentally controlled facility which was maintained at 74 __.3°F and 35-50% relative humidity, with a 12-hr light/ dark cycle. In some experiments, the rats were treated by gavage with corn oil alone, or with the indicated compounds suspended in corn oil. For enzyme induction studies, the rats were given the compounds by gavage at 50 mg/kg body weight/day for 10 consecutive days. On day 11, the rats were anaesthetized with 65 mg pentobarbital/kg body weight by ip injection. Blood samples were taken from the left ventricle by heart puncture with a heparinized syringe, and centrifuged to obtain plasma, which was stored at 4°C for no more than 48 hr. CC14 was given by ip injection, and the rats were killed after 24 hr. Liver fractions were prepared after portal vein perfusion in situ with 0.9% NaCI at room temperature. The liver was excised, placed in 0.9% ice-cold saline and trimmed of connective tissue. A 10% homogenate was prepared in ice-cold 0.1 M-potassium phosphate buffer (pH 7.4) containing 1 mM-EDTA. A 9000-g postmitochondrial supernatant fraction (Shertzer, 1984), and 105,000-g pellet (microsomes) and cytosolic fractions (Shertzer and Duthu, 1979) were prepared as described previously.

Hepatocytes. Hepatocytes were prepared by collagenase treatment as previously described (Reitman et al., 1988). In order to improve viability, cells were centrifuged through 0.508 g Percoll/ml (Pharmacia AB, Uppsala, Sweden) in 137mM-NaCI, 8.1mMNa2HPO 4 and 1.5 mM-KH2PO4 (pH 7.4). Indole compounds were added as solutions in dimethylsulphoxide (DMSO), with the final concentration of DMSO never exceeding 5#l/ml cell suspension. Where indicated, toxicants were added 5 min after the indole compound: CC14, MMS and N-methyl-N'nitro-N-nitrosoguanidine (MMNG) were added as freshly prepared solutions in DMSO, ethanol and 0.9% NaC1, respectively. Viability was determined as the percentage of cells that excluded 0.2% trypan blue. Assays. Plasma enzyme activities were determined as described in the references: alanine aminotransferase (ALT [EC 2.6.1.2], Segal and Matsuzawa, 1970); ornithine transcarbamylase (OTC [EC 2.1.3.3], Vassef, 1978); alkaline phosphatase (ALK [EC3.1.3.1], Bowers and McComb, 1975). Liver enzyme activities were assayed as described: aminopyrine N-demethylase (Shertzer, 1982); 7-ethoxyresorufin O-deethylase (Burke and Mayer, 1974); high affinity N D M A N-demethylase I (Shertzer, 1984); ascorbate synthase (L-gulonolactone oxidase [EC 1.1.3.8], Sato and Udenfriend, 1978) for which the ascorbic acid product was assayed (Zannoni et al., 1974); NADPH cytochrome c oxidoreductase ([EC 1.6.2.4], Shertzer and Duthu, 1979); UDP-glucuronosyl transferase (UDPGT [EC 2.4.1.17], Temple et al., 1971); glutathione peroxidase (GSH-Px [EC 1.11.1.9], Gfinzler and Floh6, 1985); quinone reductase ([EC 1.6.99.2], Ernster, 1967); superoxide dismutase (SOD [EC 1.15.1.1], Oberley and Spitz, 1985), cytochrome P-450 (Johannesen and DePierre, 1978). Glutathione reductase (GSSG-Red [EC 1.6.4.2]) activity was assayed at 25°C in a reaction mixture consisting of 1 mM-GSSG, 0.1 mM-Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)], 0.15 mM-NADPH, and postmicrosomal cytosol, in 0.1 M-potassium phosphate buffer containing 1 mM-ethylenediaminetetraacetic acid (EDTA) (pH 7.0, 1.5 ml). The rate of increase in absorbance due to the production of thiolate anion was determined at 410 nm, and blank reaction rates (without cytosoi) were subtracted from rates obtained with the complete mixture. GSH was used as standard to calculate #mol GSH formed/min. DNA was assayed as described by Kissane and Robin (1958) as modified by Setaro and Morley (1976). Protein was determined using the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL, USA) according to the technical procedure supplied by the manufacturer. CCl4-initiated lipid peroxidation in microsomes fortified with an NADPH regenerating system was assayed as described by Shertzer et al. (1987a). The microsomes were exposed to I mMCC14. Statistics. Data were analysed by one-way analysis of variance. When indicated by ANOVA, differences between group means were evaluated by Student's t-test (one-sided), based on the mean square residual from the analysis of variance in question. Statistics were evaluated using the Number Cruncher Statistical System (NCSS, Kaysville, UT, USA).

Chemoprotection by indole compounds in rats

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H. G. SI-IERTZER a n d M. SAINSBURY

394

Table 1. Hepatotoxicity in rats treated with CCI4 1 hr after treatment with indole compounds Plasma enzyme levels ALT (gmol/min.ml plasma)

Treatmenti" Corn oil (control)~ CCI4 I-3-C + CCI4 DHII + CCI4 THII + CCI,

OTC (U/ml plasma)

0.03 + 0.01 2.47 + 0.49 *a 1.34 4- 0.27 *a,b 0.93 4- 0.17 *a'b 0.39 4- 0.11 .=.b.¢

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12.9 + 2.9 3790 + 319 *~ 2390 4- 347 *='b 1428 4- 296 *''b'=' 704 4- 114*a'b'¢'d

11.6 ___0.8 25.9 + 3.8 *= 18.3 4- 2.3 *a'b 13.2 4- 1.9*b'¢ 12.0 4- 2.1 *b'¢

ALT = alanine aminotransferase OTC = ornithine transcarbamylase ALK = alkaline phosphatase l'Rats were treated by garage with 1.5 ml corn oil, 50 mg I-3-C, 25 mg DHII or 25 mg THII/kg body weight. The indole compounds were suspended in corn oil so that 1.5 ml of the suspension was administered/kg body weight. After 1 hr, the mice were injected ip with corn oil (2 ml/kg body weight) or CC14 (24 mg/kg body weight) in corn oil. Blood samples were taken to prepare plasma 24 hr after the CCI4 dose, and the rats were killed. :~AI1 of the values for the different vehicle control groups were the same, and therefore the results are shown only for the group treated with corn oil both by gavage and by ip injection. Values are means 4- SEM for groups of six rats. Those marked with an asterisk differ significantly (*P < 0.05) from the corresponding groups treated as follows: avehicle control; bCCI4; 'I-3-C + CCI4; dDHII + CCI4.

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Fig. 3. P a r a m e t e r s of hepatic response in rats to corn-oil vehicle (CO), indole-3-carbinol (I-3-C), p h e n o b a r b i t a l (PB), f l - n a p h t h o f l a v o n e (flNF), 5,10-dihydroindeno[1,2-b]indole ( D H I I ) or 4b,5,9b,10t e t r a h y d r o i n d e n o [ 1 , 2 - b ] i n d o l e (THII). Liver weight, D N A a n d c y t o c h r o m e P - 4 5 0 were d e t e r m i n e d experimentally; m g m i c r o s o m a l p r o t e i n was c a l c u l a t e d by d i v i d i n g n m o l c y t o c h r o m e P - 4 5 0 / g tissue (in w h o l e h o m o g e n a t e ) by n m o l c y t o c h r o m e P - 4 5 0 / m g protein (in a m i c r o s o m a l suspension). Values are m e a n s _+ S E M for six rats/group. A s t e r i s k s indicate a significant increase in the m e a n value over t h a t o f the C O controls (*P < 0.05).

Chemoprotection by indole compounds in rats RESULTS The abilities of three indole compounds to inhibit CC14-initiated lipid peroxidation in liver microsomes were compared (Fig. 1). DHII showed an IC50 of about 2/~M, equivalent to that of butylated hydroxytoluene (BHT). I-3-C was about 20-fold less effective, while THII was 10-fold more effective in inhibiting lipid peroxidation. Each of the three indole antioxidants was effective in inhibiting the decrease in hepatocyte viability caused by various toxicants (Fig. 2). For each toxicant, I-3-C was the least effective. THII was somewhat more effective than D H I I in protecting against CC14 and M N N G toxicity, while DHII was superior in the case of MMS. In order to determine whether the experiments in hepatocytes accurately reflected the situation in vivo, the indole antioxidants were administered by gavage to rats 1 hr before the administration of CC14 (Table 1). The resulting 24-hr hepatotoxicity, indicated by the magnitude of the release of tissue enzymes into the blood, was significantly reduced by each of the indole compounds,

395

with the order of efficacy THII > D H I I > I-3-C. These results indicate that it is possible that similar mechanisms of protection are operating in vivo and in vitro.

Several classes of antioxidants induce various hepatic enzymes involved in biotransformation (Prochaska and Talalay, 1988; Talalay et aL, 1988). Levels of some of these enzymes, such as quinone reductase, SOD and glutathione metabolizing enzymes, have been correlated with the onset of chemical neoplasia (Segura-Aguilar et al., 1990; Talalay et al., 1988; Wattenberg, 1985). Therefore, the enzyme induction properties of the three indole compounds was examined. After 10 days of treatment (50 mg compound/kg body weight/day) none of the indole compounds induced hepatic hypertrophy (Fig. 3), as indicated by liver weight and D N A content. Of the three indoles, only I-3-C induced a modest increase in cytochrome P-450. Although none of the indoles affected the rate of N-demethylation of N,N-dimethylaminopyrine (Fig. 4), all of the compounds examined enhanced O-deethylation of ethoxyresorufin, with the

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Fig. 4. Levels of phase I hepatic enzymes in rats after treatment with corn-oil vehicle (CO), indole-3carbinol (I-3-C), phenobarbital (PB), fl-naphthflavone (flNF), 5,10-dihydroindeno[l,2-b]indole (DHII) or 4b,5,9b,10-tetrahydroindeno[1,2-b]indole (THII). The assays were performed using the microsomal cell fraction, and the results are expressed as means + SEM for six independent determinations. An asterisk indicates a significant difference in the mean value from that of the CO control (*P < 0.05).

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Fig. 5. Liver enzyme activities in rats after treatment with corn-oil vehicle (CO), indole-3-carbinol (I-3-C), phenobarbital (PB),//-naphthflavone (~NF), 5,10-dihydroindeno[l,2-b]indole(DHII) or 4b,5,9b, lO-tetrahydroindeno[1,2-b]indole (THII). The assay for ascorbate synthase was performed using the whole liver homogenate, while NADPH cytochrome c reductase and UDP-glucuronosyl transferase assays were carried out using the microsomal fraction. Values are means + SEM for six rats/group. An asterisk indicates a significant increase in the mean value over that of the CO control (*P < 0.05).

order of efficacy being/~-naphthoflavone > I-3-C > D H I I > T H I I > phenobarbital. A very different pattern was observed for the high affinity N-demethyiation of N D M A , where none of the compounds acted as inducers, and l/-naphthoflavone and D H I I produced slight but significant decreases in this activity.

Since I-3-C has previously been shown to protect against N D M A hepatotoxicity, and to preserve hepatic ascorbate levels (Shertzer eta/., 1987b), it was thought that a possible mechanism of protection against N D M A toxicity could involve a stimulation in ascorbate synthase (L-gulonolactone oxidase). How-

Chemoprotection by indole compounds in rats

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Fig. 6. Levelsof enzymesrelated to glutathione metabolism in the livers of rats treated with corn-oil vehicle (CO), indole-3-carbinol (I-3-C), phenobarbital (PB), fl-naphthflavone (flNF), 5,10-dihydroindeno[1,2b]indole (DHII) or 4b,5,9b,10-tetrahydroindeno[l,2-b]indole (THII). The glutathione S-transferase assay was performed using the whole tissue homogenate, while the GSSG reductase and GSH peroxidase assays were performed using the 105,000-g supernatant fraction. Values are expressed as means _+SEM for six rats/group. An asterisk indicates a significant difference in the mean value from that of the CO control (*P < 0.05). ever, this does not appear to be the case, since this activity was unaltered by any of the agents examined (Fig. 5). Similarly, microsomal NADPH cytochrome c reductase was unaffected by the indole compounds. Other non-oxidative enzymes were markedly affected by the indole compounds. The activities of four enzymes were increased by I-3-C and DHII, with no increase by THII. These were U D P G T (Fig. 5),

GST and GSSG-Red (Fig. 6) and quinone reductase (Fig. 7). Two other enzymes that may be important in mitigating cellular oxidative stress are GSH-Px (Fig. 6) and SOD (Fig. 7) (Deby and Goutier, 1990; Segura-Aguilar et al., 1990). These activities were not affected by THII, but both were significantlydecreased by I-3-C. DHII decreased GSH-Px by about 20%, but had no effect on SOD activity.

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Fig. 7. Activities o f N A D ( P ) H : q u i n o n e reductase a n d s u p e r o x i d e d i s m u t a s e in the livers o f rats treated w i t h corn-oil vehicle (CO), i n d o l e - 3 - c a r b i n o l (I-3-C), p h e n o b a r b i t a l (PB), f l - n a p h t h f l a v o n e (fl N F ) , 5,10-dihydroindeno[ 1,2-b ]indole ( D H I I ) or 4b,5,gb, 1 0 - t e t r a h y d r o i n d e n o [1,2-b ]indole (THII). The N A D ( P ) H : q u i n o n e reductase assay was p e r f o r m e d using the 9000-g s u p e r n a t a n t fraction, while the s u p e r o x i d e d i s m u t a s e assay was carried o u t using the 105,000-g s u p e r n a t a n t fraction. Values are expressed as the m e a n s + S E M for six r a t s / g r o u p . A n asterisk indicates a significant difference in the m e a n value f r o m t h a t o f the C O c o n t r o l (*P < 0.05).

In an attempt to correlate the alterations in enzyme profiles with possible chemoprotection by indole compounds, rats that had been treated with I-3-C, DHII or THII for 10 days as described above, were exposed 24 hr after the last treatment to CC14, and 24hr later hepatotoxicity was estimated by

measuring plasma levels of tissue enzymes indicative of liver damage (Table 2). Surprisingly, I-3-C exacerbated CC14 toxicity, while DHII ameliorated the increase in serum enzyme levels by about 50%. THII had no apparent effect on CCI4 toxicity in this experiment.

Table 2. Hepatotoxicity in rats treated with CC14 after 10 days of treatment with indole compounds Plasma enzyme levels Treatmentt Corn oil (control):[: CCI4 I-3-C + CCl4 DHII + CCI4 THII + CCI 4

ALT (/~mol/min. ml plasma) 0.02 + 0.01 2.22 + 0.47 *~ 3.18 + 0.44 *='b 1.04 _+0.29 *='b': 2.16 + 0.49 *='d

OTC (U/ml plasma) 15.6 _+4.1 3260 _+ 510 *a 4370 + 520 *=,b 1260 _+ 350 *a'b'c 3040 + 510 *='d

ALK (#tool/rain .ml plasma) 12.5 _+ 1.2 23.2 _+ 2.9 *= 28.6 __ 1.8*~'b 15.6 + 1.6*~'b'c 25.0 + 3.2 *a'd

ALT = alanine aminotransfcras¢ OTC = ornithine transcarbamylase ALK = alkaline phosphatase 1"Rats were treated by gavage for 10 consecutive days with 1.5 ml corn oil, 50 mg I-3-C, 50 mg DHII or 50 mg THII/kg body weight. The indole compounds were administered in corn oil, and 24 hr after the last treatment the rats were given 24 mg CCI4 or 2 ml corn oil/kg body weight by ip injection. After 24 hr, blood samples were obtained and the rats were killed. ~:AII of the values for the different vehicle control groups were the same. Therefore, only the corn-oil garage plus corn-oil ip injection data arc presented. Values arc means + SEM for groups of six rats. Those marked with an asterisk differ significantly (*P < 0.05) from the corresponding groups treated as follows: "vehicle control; bCCI4; ¢I-3-C + CCI4; dDHII + CCI(.

Chemoprotection by indole compounds in rats DISCUSSION

The potential value of I-3-C as a chemoprotective agent against chemical toxicity and carcinogenesis has been under investigation for some time. The compound is a natural component of the human diet (Loub et al., 1975), acts as a scavenger for free radicals (Shertzer et al., 1988) and reactive electrophiles (Shertzer and Tabor, 1988), and stabilizes biological membranes against fluidity changes (Shertzer et al., 1991). However, I-3-C is toxic, as indicated by its potential to enhance carcinogenic promotion in mouse skin (Birt et al., 1986), deplete hepatic glutathione levels, and produce hepatic toxicity and neurological impairment (Shertzer and Sainsbury, 1991). It has been suggested that protection against chemical carcinogenesis is sometimes correlated with the ability of some chemoprotectants to induce a specific set of non-oxidative enzymes, including GST, UDPGT and quinone reductase (Nebert, 1989; Nebert et al., 1990; Talalay et al., 1988; Wattenberg, 1985). In mice, the induction of these enzymes is partly under the control of the [Ah] gene locus. Therefore, TCDD and other [Ah] inducers also increase oxidative enzyme activities catalysed by the CYP1AI and CYPIA2 forms of cytochromes P-450 (Nebert, 1989; Nebert et al., 1990). These inducers of both oxidative and non-oxidative enzymes have been termed bifunctional, whereas those agents that induce only nonoxidative enzymes have been termed monofunctional (Prochaska and Talalay, 1988; Talalay et al., 1988). These authors suggest that monofunctional agents are preferable for potential chemoprotective compounds, because they observed correlations between the abilities of compounds to act as monofunctional enzyme inducers, Michael reaction acceptors, and inhibitors of some types of chemical carcinogenesis. In the present study, I-3-C behaved as a bifunctional enzyme inducer, DHII as a monofunctional inducer (very weak oxidative component), and THII as a non-inducer. The inability of a chemical to induce any oxidative or non-oxidative metabolism may be desirable, since unforeseen complications may result from the metabolic activation of some xenobiotic to which the animal may be exposed. An attempt has been made to discriminate between the chemoprotection that is afforded by indole compounds as a result of changes in enzyme activity, and those protective effects that are not the result of such changes (i.e. direct effects such as radical scavenging or membrane stabilization). One major difficulty with such an analysis is that a detailed pharmacokinetic profile has only been obtained for I-3-C in mice (Shertzer et al., 1987a). In this case the 50% clearance time from the blood was about 6 hr. Since the mice were killed 24hr after the last enzyme-inducing dosage of I-3-C, little compound would be available to participate directly in modulating CCI 4 toxicity. If the increased susceptibility of rats to CCl4-mediated hepatotoxicity after 10 days of treatment with I-3-C were related to changes in enzyme activities, then the 55°/'o reduction in SOD activity could be indicative of the expression of this free-radical mediated toxic response. Such speculation may not be made for DHII in the absence of pharmacokinetic data. On the other hand, protection against CC14 hepatotoxicity FCT

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by THII does not appear to be related to enzyme induction, since protection is lost 24 hr after the last THII treatment. It appears that the double bond between the indole and indene moieties of DHII is necessary for the enzyme inducing properties of this compound, since THII (saturated at this site) is not an inducing agent. It has previously been suggested that Michael reaction acceptors such as enones, which consist of an electron-withdrawing substituent in conjugation with an olefinic bond, may act as inducers of the [Ah] gene locus (Talalay et al., 1988). Indoles such as DHII might behave similarly, even though they are polarized in the opposite direction to enones. Acting as enamines, these compounds may undergo electrophilic attack at the t - c a r b o n atom (e.g. protonation), followed by the capture of a potential nucleophile at the or-position. These reactions are typical of indoles and related compounds (Livingstone, 1973). In the case of I-3-C, unidentified reaction product(s) with [Ah] gene locus-inducing properties appear to be formed under acid conditions (Bradfield and Bjeldanes, 1987), such as occur in the stomach. This may explain the poor ability of I-3-C to induce aryl hydrocarbon hydroxylase when administered ip (Shertzer, 1982), in contrast to its relative potency following oral administration (Bradfield and Bjeldanes, 1987; Shertzer, 1982; Shertzer and Sainsbury, 1991), since I-3-C itself does not bind to the TCDD receptor in order to induce enzymes associated with the [Ah] gene battery. We conclude that DHII and THII have potential for further development as chemoprotective or therapeutic agents in toxicological, carcinogenic or disease processes. Either DHII by virtue of its enzyme induction properties, or THII by virtue of superior antioxidant efficacy, may exhibit better protection in a given situation. Acknowledgements--Part of this investigation was supported by USPHS grant ES-03373. The synthesis of DHII was supported by the Cancer Research Campaign (UK). The authors thank Ms Venita Grant for typing this manuscript.

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