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Effects of soya bean flakes and liquorice root extract on enzyme induction and toxicity in B6C3F1 mice
Fd Chem. Toxic. Vol. 31, No. 5, pp. 343-350, 1993 Printed in Great Britain. All rights reserved
0278-6915/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
EFFECTS OF SOYA BEAN FLAKES AND LIQUORICE ROOT EXTRACT ON ENZYME INDUCTION AND TOXICITY IN B6C3FI MICE J. C. MIRSALIS*,C. M. HAMILTON,J. E. SCHINDLER,C. E. GREEN and J. E. DABBS Toxicology Laboratory, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493, USA (Accepted 20 December 1992)
Abstract--Both soya bean flakes (SBF) and liquorice root extract (LRE) have previously been reported to have anticarcinogenic properties, which have been thought to be related to an increased activity of specific enzymes responsible for the detoxification of chemical carcinogens. 30- and 90-day studies were conducted in male B6C3F~ mice to determine which, if any, of several detoxification enzymes are induced by SBF or LRE. Mice fed 8 and 25% LRE showed a variety of adverse clinical signs, poor weight gain and 30% mortality. Significant increases in liver:body weight ratios were observed in both the SBF and LRE groups. No significant treatment-related gross autopsy findings were observed in any of the SBF groups. A number of abnormalities were observed in the LRE groups, including lesions of the kidney, liver, spleen and thymus. Liver samples from the 90-day study were analysed for 7-ethoxycoumarin O-deethylase (7-ECOD), benzo[a]pyrene hydroxylase (BPH), superoxide dismutase (SOD), glutathione S-transferase (GST) and UDP-glucuronyl transferase (UDPGT) at 90 days, and at an interim 30-day autopsy. No treatment-related increases were observed for BPH or SOD. Both SBF and LRE induced modest increases in UDPGT activity. SBF induced modest increases in GST activity, but LRE decreased this activity. 7-ECOD activity was significantly increased by LRE and decreased by SBF. Samples from a 30-day study in which both LRE and SBF were administered at various dose levels were examined for UDPGT activity; all dose groups showed decreases in UDPGT activity relative to controls. The results suggest that both SBF and LRE may alter the activities of specific enzymes involved in the detoxification of chemical carcinogens; however, the combination of these two foodstuffs may not produce an additive effect in B6C3F t mice.
INTRODUCTION Considerable evidence now suggests that diet plays a major role in modulating the effects of chemical carcinogens, and a wide variety of dietary components have been suggested to possess the ability to prevent the formation of tumours (Wattenberg, 1985). This 'chemopreventive' activity is observed for many naturally occurring foods, and foods that possess anticarcinogenic properties frequently share a c o m m o n attribute: they induce a variety of enzymes involved in the detoxification and excretion of carcinogenic and toxic chemicals (Williams, 1971). Understanding the effects of specific foods, separately and in combination, on enzyme induction or inhibition will provide valuable information on the mechanism of chemoprevention of specific foods. Both soya bean and liquorice have shown chemopreventive properties in a variety of models. Soya beans contain a variety of biologically active compounds; the most intensively studied have been
*To whom correspondence should be addressed. Abbreviations: BPH = benzo[a]pyrene hydroxylase; 7ECOD = 7-ethoxycoumarin O-deethylase; GCA = glycyrrhetinic acid; GCZ = glycyrrhizin; GST = glutathione S-transferase; LRE = liquorice root extract; SBF = soya bean flakes; SOD = superoxide dismutase; UDPGT = UDP-glucuronyl transferase.
the isoflavones (Naim et aL, 1974) and the protease inhibitors (Birk, 1985). Several studies have evaluated the chemopreventive effects of soya bean, but most have evaluated the effects of purified protease inhibitors. Raw soya beans or protease inhibitors have been demonstrated to be effective chemopreventive agents in a variety of animal models (Barnes et al., 1983; Mokhtar et al., 1988; Witschi and Kennedy, 1989), and limited epidemiological data also suggest a protective effect of soya bean in humans (Hirayama, 1982 and 1985). Autoclaving or chemical processing of soya beans destroys much of the protease inhibitor activity, along with the associated chemopreventive effects. Raw soya flour is capable of decreasing the pancreatic carcinogenicity of N-nitrosobis-(2-oxopropyl)amine in Syrian golden hamsters, whereas heated soya flour was ineffective in decreasing the tumour incidence (Hasdai and Liener, 1985). Protease inhibitots have been reported to cause pancreatic lesions in rats (Gumbmann et al., 1989; Hasdai and Liener, 1985; McGuinness et al., 1987; McGuinness and Wormsley, 1986; Morgan et al., 1977), but this effect does not occur in mice (Hasdai and Liener, 1986; Weed et al., 1985). Little information is available on the induction of enzymes by specific soya bean flavones, but information is available on the effects of other structurally 343
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J.C. MIRSALISet al.
related compounds. The flavones are classic enzyme inducers that affect a variety of phase I and phase II biotransformation pathways (Bruschi and Priestly, 1988; Dragani et al., 1983; Miller and Halpert, 1987). Hepatic lipogenic enzyme induction, fatty acid synthesis and triglyceride levels were all markedly lower in rats fed soya bean protein than in those fed casein (Iritani et al., 1988). The main biologically active component of liquorice is the triterpene glycyrrhizin (GCZ), which is a diglucuronide of glycyrrhetinic acid (GCA). In addition to GCZ and GCA, liquorice contains a variety of flavones, with the structures and content varying with the species and region of growth (Demizu et al. 1988; Hatano et al., 1988; Okada et al., 1989). GCA is a weak inhibitor of the hepatic microsomal hydroxylation of aniline and acetanilide in vitro (Leibman, 1971). GCA, methyl-GCA and GCA-diacetate prevent elevation of serum alanine aminotransferase and aspartate aminotransferase during inflammatory reactions (Tangri et al., 1965). In the present study, the effects were evaluated of processed soya bean flakes (SBF) and liquorice root extract (LRE) on the activity of several phase I and phase II detoxification enzymes in male B6C3F t mice. In addition, a variety of effects were reported of the two foodstuffs on weight gain, survival and other clinical signs. A 90-day feeding study was conducted to determine the effects of SBF and LRE administered separately. A 30-day study was then conducted in which SBF and LRE were administered in combination. The purpose of this 'synergism' study was to determine whether the two food products could produce an additive or synergistic effect on the enzyme induction observed in the 90-day study.
MATERIALS AND METHODS
Animals. Care and housing of all animals was carried out in a facility accredited by the American Association for Accreditation of Laboratory Animal Care. The 1970 Animal Welfare Act and its amendments (P.L. 89-544 and P.L. 91-579), and the principles promulgated by the National Institutes of Health (NIH Publication No. 85-23) in the Guide for the Care and Use of Laboratory Animals were adhered to at all times. Male B6C3F~ mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA, USA) and were approximately 6 wk old at the initiation of the study. Mice were individually housed in polycarbonate solid-bottomed suspended drawer-type cages containing Sani-Chips hardwood bedding (P. J. Murphy Forest Products, Montville, N J, USA). The animals were provided with deionized UV-exposed water ad lib. The animal room was maintained at 72 _+ 3°F, with a relative humidity ranging from 30 to 65% and a 12-hr light/dark cycle throughout the study. The mice were quarantined and acclimatized for 10 days before the
start of dosing. Each dose group contained five male mice in both the 90- and 30-day studies. Diets. Mice received a semi-purified diet (modified AIN-76A) consisting of 20% casein, 0.3% DL-methionine, 52% corn starch, 13% dextrose, 5% corn oil, 5% alphacel, 3.5% AIN-76 mineral premix, 1% AIN-76 vitamin premix and 0.2% choline bitartrate (American Institute of Nutrition, 1977 and 1980). All diets were in meal form and were purchased from Zeigler Bros (Gardners, PA, USA). SBF was provided by Central Soya (Fort Wayne, IN, USA; Lot RRSBM900011), and consisted of toasted flaked soya beans; this heating destroyed any protease inhibitor activity. LRE was provided by MacAndrews & Forbes Co. (Camden, NJ, USA; Lot AHLIC900004-10) and consisted of a mixture (50: 50) of powdered liquorice root from Russian and Chinese liquorice. The same batch of LRE and SBF was used for all studies. Test diets containing SBF, LRE or both, were prepared by mixing ground material into the AIN-76A diet with a twin-shelled V-blender (Patterson-Kelly Co., East Strodsburg, PA, USA). All test diets were adjusted for the loss of minerals, vitamins, choline bitartrate and DL-methionine displaced by the addition of the test compounds. Analytical chemistry studies demonstrated that the diets were stable for at least 6 wk, and homogeneity studies revealed that the coefficient of variation was less than 5 0 . For the 90-day study, animals received 0.8, 2.5, 8 or 25% SBF or LRE. For the 30-day synergism study, mice received all possible combinations of 20, 8 and 2.5% SBF, and 5, 2.5 and 0.8% LRE. Pair-fed control groups. Pair-fed control groups were added to the 90-day study; this corresponded to the make-up of the 25% SBF and LRE groups. Mice fed the highest dose of LRE showed considerable weight loss and unwillingness to eat the test diet; therefore, from wk 3 the LRE pair-fed controls were matched to the next highest dose group (8% LRE) for the remainder of the study. The pair-fed control diet consisted of AIN-76A diet with casein and dextrose added to match the protein and carbohydrate levels found in the diets containing the highest doses of SBF and LRE. The SBF pair-fed control diet consisted of 25% protein, 4.5% corn oil, 5% vitamins/minerals and 65.6% carbohydrate. The LRE pair-fed control diet consisted of 17% protein, 6.6% fat, 5% vitamins/minerals and 71.4% carbohydrate. AIN-76A diet was fed to a control group, which was allowed food ad lib. In the 30-day study, a matched diet containing the protein and carbohydrate levels of the highest SBF/LRE dose groups was prepared but was given ad lib to the mice. In-life measurements. Body weights and food co sumption were determined weekly throughout the study. Mice were observed daily throughout both studies for signs of toxicity including evaluation of appearance, posture, gait and behaviour, as well as any abnormalities in respiration and body
Enzyme induction by soya bean and liquorice
345
Table 1. Body weights and liver: body weight ratios of mice after 30 or 90 days of treatment with liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days 90 days Dose No. of Body weight No. of Body weight Treatment (%) animals (g) Liver/body animals (g) Liver/body Control 0.0 5 34.0 _+ 1.1 4.74 -+ 0.26 5 43.6 _+2.9 5.37 _+0.35 Pair-fed (SBF) SBF
0.0 0.8 2.5 8.0 25.0
Pair-fed (LRE) LRE
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
31.5 _+2.1 35. I _+2.4 33.3_+1.9 32.9 _+ 1.7 32.5_+1.7
5 31.9 _+2.1 5 32.8 + 1.5 5 28.3_+2.0 5 22.2 _+2.7* 3 24.4 __.0.4* Liver/body = 100 x liver *Lower than pair-fed control (P < 0.01; Student's t-test). "t'Greater than pair-fed control (P < 0.05; Student's t-test). :~Greater than pair-fed control (P < 0.01; Student's t-test). Values are means + SD of the numbers of mice indicated.
t e m p e r a t u r e o r u r i n e a n d faeces. All e x t e r n a l s t r u c t u r e s were e x a m i n e d a n d e a c h m o u s e w a s p a l p a t e d f o r e x t e r n a l m a s s e s . A c o m p l e t e a u t o p s y w a s perf o r m e d o n all m i c e f o l l o w i n g killing b y C O : i n h a l a tion. A complete autopsy included external e x a m i n a t i o n o f all b o d y s u r f a c e s a n d orifices, a n d o f all o r g a n s a n d t i s s u e s o f t h e a b d o m i n a l , t h o r a c i c a n d c r a n i a l cavities. L i v e r s were r e m o v e d , w e i g h e d a n d used for the evaluation of enzymes. Enzyme activity. D u r i n g a u t o p s y , t h e liver w a s e x c i s e d f r o m t h e a n i m a l a n d w e i g h e d in a c o v e r e d b e a k e r . T h e t i s s u e w a s m i n c e d , a d d e d to chilled 0.15 M-KCI b u f f e r (3 m l / g liver) a n d h o m o g e n i z e d w i t h a P o l y t r o n h o m o g e n i z e r f o r 30 sec o r less. T h e h o m o g e n a t e w a s c e n t r i f u g e d at 9 0 0 0 g f o r 15 m i n at 4°C. The supernatant containing microsomes and soluble p r o t e i n s w a s collected, a l i q u o t e d a n d s t o r e d f r o z e n a t -135°C until assayed. Each sample was measured for protein content by the spectrophotometric assay o f B r a d f o r d (1976) u s i n g C o o m a s s i e Blue dye. All b i o c h e m i c a l a s s a y v a l u e s were n o r m a l i z e d u s i n g t h e p r o t e i n c o n t e n t d a t a . Specific e n z y m e s were e v a l u a t e d
4.21 _+0.23 4.98 _+0.26~, 4.75 -+ 0.26I" 4.54 _+0.29 4.94 -+ 0.21~ 4.84 _+0.47 5.06 _+0.34 5.53_+0.67 6.60 _+0.32~ 5.82 _+0.31t weight/body weight
5 5 5 5 5
43.2 _+ 1.3 44.5 _+ 1.2 43.5_+2.2 45.3 -t- 2.4 42.0_+1.3
5.01 _+0.46 5.27 __.0.34 5.10_+0.16 5.21 ___0.21 4.90_+0.13
5 5 5 2 4
43.8 _+ 1.3 39.9 _+4.0 32.8_+2.1" 27.8 _+3.3 27.5 _+ 1,4"
4.90 _+0.27 5.22 _+0.32 6.46 -+ 0.30:~ 9.54 _+ 1.93 7.99 _+0.88~
as d e s c r i b e d below. D e e t h y l a t i o n o f 7 - e t h o x y c o u m a r i n w a s d e t e r m i n e d by m e a s u r i n g p r o d u c t formation using fluorescence spectroscopy. Tissue h o m o g e n a t e s a n d c o f a c t o r s were i n c u b a t e d as des c r i b e d b y G r e e n l e e a n d P o l a n d (1978). B e n z o [a]pyrene hydroxylase (BPH) was measured using the r a d i o a c t i v i t y a s s a y d e v e l o p e d b y D e P i e r r e et al. (1975). Glutathione S-transferase (GST) was m e a s u r e d in tissue h o m o g e n a t e s u s i n g 1-chloro-2,4d i n i t r o b e n z e n e as d e s c r i b e d b y H a b i g et aL (1974). U D P - g l u c u r o n y l t r a n s f e r a s e ( U D P G T ) activity w a s d e t e r m i n e d b y m e a s u r i n g t h e d i s a p p e a r a n c e o f 4n i t r o p h e n o l ( L u c i e r et al., 1977). S u p e r o x i d e d i s m u tase (SOD) was measured continuously with an o x y g e n electrode, w h i c h m e a s u r e s t h e rate o f o x y g e n consumption induced by the photochemical generation of superoxide from dissolved oxygen followed b y d i s m u t a t i o n to o x y g e n a n d h y d r o g e n p e r o x i d e ( M a r s h a l l a n d W o r s f o l d , 1978). Statistical a n a l y s e s were c a r r i e d o u t u s i n g S t u d e n t ' s t-test, a n d a difference at P ~< 0.05 w a s c o n s i d e r e d to be significant.
Table 2. Protein contents of mouse liver homogenates after 30 or 90 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days 90 days Dose No. of No. of Treatment (%) animals mg/ml* mg/gt animals mg/ml* mg/gt Control 0.0 5 20.2 + 1.1 60.7 + 3.3 5 20.7 + 1.7 62.0 + 5.0 Pair-fed (SBF) SBF SBF SBF SBF
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
Pair-fed (LRE) 0.0 5 LRE 0.8 5 LRE 2.5 5 LRE 8.0 2 LRE 25.0 4 *mg protein per ml S-9 (mean _+SD). tmg protein per g wet tissue (mean _+SD).
23.0_+2.1 18.7 _+2.1 20.5 _+ 1.7 19.8+2.2 20.9 _+2.6
69.1 _+6.4 56.2 _+6.4 61.4 _+5.2 59.3_+6.5 62.8 _+7.9
5 5 5 5 5
23.0_+ 1.8 19.6 _+4.0 19.5 _+0.9 17.3+ 1.0 19.9 _+ 1.4
69.1 _+5.3 58.7 _+ I 1.9 58.4 _+2.8 52.0_+3.1 59.6 _+4.3
20.0 _+2.5 22.0 _+ 1.8 23.1_+1.9 23.0 _+0.6 22.3 _+2.3
60.0 _+7.6 66.0 _+5.4 69.2_+5.8 68.9 _+ 1.9 66.8 _+6.8
5 5 5 5 3
21.5 _+2.0 20.9 _+0.9 21.8_+0.9 24.4 _+ 1.2 22.3 _+ t.5
64.5 _+5.9 62.8 _+2.7 65.4_+2.7 73.1 _+3.6 66.8 _+4.6
J. C. MIRSALIS et al.
346
Table 3. Benzo[a]pyrene hydroxylase (BPH) and superoxide dismutase (SOD) activities in the livers of mice after 30 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) BPH Dose (%)
No. of animals
Activity*
0.0
5
2.5 + 1.4
Pair-fed (SBF) SBF SBF SBF SBF
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
Pair-fed (LRE) LRE LRE LRE LRE
0.0 0.8 2.5 8.0 25.0
5 5 5 5 3
Treatment Control
SOD % Pair-fed
No. of animals
Activity*
--
5
1.24 _+ 0.65
--
2.2 + 0.9 1.8 + 0.6 2.4 + 0.8 2.5 _+ 1.0 2.3 + 0.9
100 70 109 114 105
5 5 5 5 5
0.92 + 0.79 1.43 + 0.58 1.58 + 0.39 1.29 _+0.97 0.82 +_ 0.54
100 155 172 140 89
1.7 + 1.8 ± 2.0 ± 3.1 ± 2.1 +
100 106 118 182 124
5 5 5 5 3
1.86 _+0.91 1.92 ± 1.00 0.44 + 0.53 2.12 ± 0.48 2.48 + 0.75
100 103 24t 114 133
1.2 0.9 1.6 1.2 0.4
% Pair-fed
*Enzyme activity (nmol/mg protein/min; mean _+ SD). tLower than control (P < 0.05; Student's t-test).
RESULTS
90-day study A 90-day study was conducted to determine which, if any, of several detoxification enzymes are affected by SBF or LRE administered individually in the diet. A 30-day interim autopsy was also included. The main clinical finding was poor weight gain in all mice given LRE except the lowest dose group (i.e. 0.8% LRE). Weight losses were observed in the first week and coincided with decreased food consumption during the same period. The 25% SBF group seemed to gain weight more slowly than the other SBF groups, but these differences were not significant when compared with pair-fed controls. Decreased body weights (v. pair-fed controls) were observed in the LRE groups at both 30- and 90-day autopsies (Table 1). Significant increases in liver: body weight ratios were observed in both the SBF and LRE groups at 30 days, but only at 90 days in the LRE groups. In the SBF groups, significant increases were observed at 30 days in both the absolute liver weight and liver:body weight ratio relative to pair-fed controis, but these increases were not dose related. In the
LRE groups significant increases in the absolute liver weight were not observed; however, when liver weights were adjusted for the lower increases in body weight liver-to-body weight increases were significant at the two highest doses after 30 days of treatment and at the highest and third highest doses after 90 days of treatment. The protein concentration of S-9 fractions prepared from the livers did not vary significantly from the control (Table 2). No adverse clinical effects were observed in SBF mice at either the 30- or 90-day autopsy. Animals in both the 8 and 25% LRE groups showed adverse clinical signs throughout the study including thin, hunched posture, a lump (0.5-1.0 cm) near the prepuce, alopecia on the abdomen and between the anus and the base of the tail, irritated swollen and red area between the anus and the base of the tail, rough fur, and lethargic, depressed and weak behaviour. Of 10 mice per group maintained on LRE, three in the 25% group and three in the 8% group were found dead or moribund before the scheduled autopsy. Deaths occured during wk 4 and 11 for the 25% LRE group and during wk 11 and 12 for the 8% LRE group. On the basis of the expected poor survival rate
Table 4. 7-Ethoxycoumarin O-deethylase activity in the livers of mice after 30 or 90 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days % Pair-fed
Activity*
--
5
1.33 _+ 0.64
--
0.49 0.19 0.15 0.48 0.42
100 42§ 52§ 67:~ 705
5 5 5 5 5
1.28 J- 0.23 1.22 + 0.13 1.18 ± 0.32 1.23 ± 0.38 1.08 ± 0.44
100 95 92 96 84
1.68 ± 0.60 2.45 + 0.36 2.59 _+0.52 3.49 + 0.80 4.29 ± 0.60
100 146 154 208t 255t
5 5 5 2 4
0.94 + 1.48 + 1.86 + 1.71 + 2.55 +
100 157 198t 182 271
No. of animals
Activity*
0.0
5
2.62 + 0.54
Pair-fed (SBF) SBF SBF SBF SBF
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
3.05 ± 1.29 + 1.59 + 2.03 + 2.13 ±
Pair-fed (LRE) LRE LRE LRE LRE
0.0 0.8 2.5 8.0 25.0
5 5 5 5 3
Treatment Control
90 days No. of animals
Dose (%)
*Enzyme activity (nmol/mg protein/min; mean + SD). "tGreater than control (P < 0.05; Student's t-test). ~Lower than control (P < 0.05; Student's t-test). §Lower than control (P < 0.01; Student's t-test).
0.28 0.39 0.43 0.33 1.01
% Pair-fed
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Table 5. UDP-Glucuronyl transferase activity in the livers of mice after 30 or 90 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days 90 days Dose No. of No. of Treatment (%) animals Activity* % Pair-fed animals Activity* % Pair-fed Control 0.0 5 1.39 + 0.55 -5 0.63 + 0.27 -Pair-fed (SBF) 0.0 5 1.18 + 0.53 SBF 0.8 5 0.91 + 0.47 SBF 2.5 5 1.47 _+0.34 SBF 8.0 5 1.20 + 0.56 SBF 25.0 5 1.67 _+0.51 Pair-fed (LRE) 0.0 5 1.14 + 0.47 LRE 0.8 5 1.11 __.0.42 LRE 2.5 5 1.39 _+0.68 LRE 8.0 5 1.81 + 0.36 LRE 25.0 3 1.27 + 0.51 *Enzyme activity (nmol/mg protein/min; mean + SD). tGreater than control (P < 0.05; Student's t-test).
in t h e 2 5 % L R E g r o u p , f o o d c o n s u m p t i o n in t h e p a i r - f e d c o n t r o l g r o u p f o r L R E w a s m o d i f i e d to m a t c h t h a t in t h e 8 % g r o u p , r a t h e r t h a n t h a t in t h e 2 5 % g r o u p , at t h e b e g i n n i n g o f w k 3. All S B F - f e d mice survived until their scheduled autopsies. N o s i g n i f i c a n t f i n d i n g s were o b s e r v e d in a n y o f t h e S B F g r o u p s , e x c e p t a single m o u s e in t h e l o w e s t d o s e g r o u p e x h i b i t i n g a m i l d l y e n l a r g e d b l a d d e r at t h e 3 0 - d a y a u t o p s y . T h i s effect w a s n o t o b s e r v e d in t h e 9 0 - d a y s t u d y a n d is n o t c o n s i d e r e d to be t r e a t m e n t related. N o s i g n i f i c a n t f i n d i n g s were o b s e r v e d in t h e t w o l o w e s t L R E d o s e g r o u p s at 30 d a y s , b u t a n u m b e r o f a b n o r m a l i t i e s were o b s e r v e d in t h e t w o h i g h e s t L R E d o s e g r o u p s . In p a r t i c u l a r , f o u r o f five m i c e in t h e 8 % L R E g r o u p a n d o n e o f t h e t h r e e s u r v i v i n g m i c e in t h e 2 5 % g r o u p h a d a b n o r m a l k i d n e y s . O t h e r a b n o r m a l i t i e s o b s e r v e d in t h e t o p t w o LRE dose groups included small thymus, enlarged s p l e e n a n d m o t t l e d liver. T h e s e f i n d i n g s were c o n f i r m e d at t h e 9 0 - d a y a u t o p s y a n d also e x t e n d e d to i n d i v i d u a l a n i m a l s in t h e 2 . 5 % d o s e g r o u p .
Enzyme assays Benzo[a]pyrene hydroxylase. A t 30 d a y s , B P H w a s i n c r e a s e d in m i c e g i v e n 8 % L R E relative to b o t h t h e
100 77 125 102 142 100 97 122 159 111
5 5 5 5 5 5 5 5 2 4
0.87 + 0.26 1.10 + 0.27 1.25 + 0.38 1.33 + 0.23 1.45 + 0.36 0.93 + 0.27 1.14 ___0.51 0.72 +..+_0.44 1.11 + 0.43 1.10 +_0.39
100 126 144 153t 167t 100 123 77 119 118
p a i r - f e d ( 1 8 2 % ) a n d ad lib. ( 1 2 4 % ) c o n t r o l s ( T a b l e 3). B e c a u s e o f c o n s i d e r a b l e a n i m a l - t o - a n i m a l varia t i o n , this i n c r e a s e w a s n o t s i g n i f i c a n t b u t is believed to be a t r u e b i o l o g i c a l increase. N o c h a n g e s in B P H activity were i n d u c e d b y S B F at 30 d a y s . T h e e n z y m e activity w a s n o t e v a l u a t e d a f t e r 90 d a y s o f t r e a t m e n t . Superoxide dismutase. C o n s i d e r a b l e a n i m a l - t o a n i m a l v a r i a t i o n w a s o b s e r v e d in this a s s a y ( T a b l e 3). Livers from some animals had no measurable SOD activity; this lack o f r e s p o n s e w a s c o n f i r m e d for s e v e r a l s a m p l e s o n s e p a r a t e d a y s . T h e e n z y m e activity m a y h a v e b e e n lost d u e to s a m p l e p r o c e s s i n g , b u t this s e e m s u n l i k e l y since all s a m p l e s were p r o c e s s e d together. T h e r e s u l t s were h i g h l y v a r i a b l e a n d t h e r e f o r e c o n c l u s i o n s a r e difficult to d r a w . T h e 2 5 % L R E g r o u p s h o w e d a n i n c r e a s e in S O D activity o v e r p a i r - f e d ( 1 3 3 % ) a n d ad lib. ( 2 0 0 % ) c o n t r o l s ; h o w ever, t h e e n z y m e activity in L R E p a i r - f e d c o n t r o l s itself w a s h i g h e r ( 1 5 0 % ) t h a n t h a t in ad lib. c o n t r o l s . Likewise, t h e 0.8, 2.5 a n d 8 % S B F g r o u p s s h o w e d i n c r e a s e s in S O D activity o v e r p a i r - f e d c o n t r o l s . In m i c e t r e a t e d w i t h L R E , t h e o n l y s i g n i f i c a n t differe n c e w a s a d e c r e a s e in S O D activity in t h e 2 . 5 % g r o u p . I n this g r o u p , t h r e e o f five a n i m a l s h a d n o o r very low activity; this m a y n o t r e p r e s e n t a significant
Table 6. Glutathione S-transferase activity in the livers of mice after 30 or 90 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days 90 days Dose No. of No. of Treatment (%) animals Activity* % Pair-fed animals Activity* % Pair-fed Control 0.0 5 101 _+ 14 -5 98 + 12 -Pair-fed (SBF) SBF SBF SBF SBF
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
90 + 12 113 + 16 117 _+9 102 _+ 13 100 + 12
Pair-fed (LRE) 0.0 5 107 __.27 LRE 0.8 5 104 + 22 LRE 2.5 5 95 __.19 LRE 8.0 5 78 + 12 LRE 25.0 3 90 __.10 *Enzyme activity (nmol/mg protein/min; mean _+SD). tGreater than control (P < 0.05; Student's t-test).
100 126 130"I" 113 I 11
5 5 5 5 5
84 +_ 15 93 + 13 96 + 10 94 + 13 99 + 16
100 III 114 112 118
100 97 89 73 84
5 5 5 2 4
100 + 90 + 87 + 91 + 95 +
100 90 87 91 95
9 10 7 2 10
J. C. MIRSALIS et al.
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Table 7. Body weights, liver: body weight ratios and UDP-glucuronyl transferase (UDPGT) activity in the livers of mice after 30 days of exposure to liquorice root extract (LRE) and processed soya bean flakes (SBF) in combination % in diet LRE SBF None NoneS" 0.8 0.8 0.8 2.5 2.5 2.5 5.0 5.0 5.0
None None12.5 8.0 20.0 2.5 8.0 20.0 2.5 8.0 20.0
No. of animals
Body weight
Liver/body
5 5 5 5 5 5 5 5 5 5 5
23.7 + 1.1 26.0 4. 1.7:~ 26.4 4. 2.0:[: 27.2 4. 1.0~ 29.9 4. 3.4:~ 27.3+1.1§ 29.3 4. 0.8§ 28. I __. 1.4~ 23.64. 1.9 23.9 + 1.3 24.4 +_ 2.2
5.89 + 0.89 5.68 4. 0.50 7.44 _ 0.445 7.59 4. 0.53§ 5.89 4. 0.21 7.06 +- 0.52~ 7.08 4. 0.35:~ 6.68 + 0.22 7.16 __ 0.81~ 7.00 __.0.98 6.85 4. 0.80
UDPGT Activity* % Control 1.70 4. 0.66 2.13 4. 0.55 1.30 + 0.19 1.04 4. 0.36 0.79 4. 0.24 1.124.0.49 0.44 4. 0.31 0.70 + 0.34 0.96+_0.18 1.28 +_0.46 1.20 +_0.31
100 125 76 61 4611 66 2611 4111 56 75 71
*Expressed as nmol/mg protein/min. t A second control group was formulated to have equivalent levels of protein, carbohydrate and lipid as in the 5% LRE/20% SBF group and the diet was provided ad lib. ~Greater than control (P < 0.05; Student's t-test). §Greater than control (P < 0.01; Student's t-test). IILowerthan control (P < 0.05; Student's t-test). Values are means + SD of the numbers of mice indicated.
effect of LRE since a consistent dose-related effect was not observed. SOD activity was not evaluated at 90 days. 7-Ethoxycoumarin O-deethylase. No increase in 7-ECOD activity was observed for the SBF groups at 30 days; in fact, this activity was significantly lower in all SBF-fed groups than in both the SBF pair-fed and ad lib. control groups (Table 4). Slight decreases in the activity (84-96% of pairfed controls) were also observed at 90 days; however, these decreases were less pronounced and not significant. At 30 days, the LRE groups showed doserelated elevations in 7-ECOD activity over LRE pair-fed controls. At the highest dose, the activity was approximately 2.5-fold higher than in pairfed controls. The increase in 7-ECOD activity was significant at the top two doses when compared with pair-fed controls, and at the top dose only when compared with ad lib. controls. The results at 90 days were comparable with those seen at 30 days, although there were fewer mice in the two highest dose groups (only two and four surviving mice at 8 and 25% LRE, respectively); therefore, a significant difference was not observed. Dose-related increases in 7-ECOD activity (approx. two- to three-fold over pair-fed controls) were observed in all LRE-treated groups. UDP-glucuronyl transferase. Slight increases in UDPGT activity were observed at 30 days in both the 25% SBF 0 4 2 % of pair-fed controls; 121% ofadlib. controls) and 8% LRE groups (150% of pair-fed controls; 129 % of ad lib. controls), but these increases were not significant (Table 5). Increases in the enzyme activity were observed in SBF animals at 90 days and were significant at the top two doses (53 and 67% over pair-fed controls, respectively). U D P G T was selected as the single endpoint for the synergism study. Glutathione S-transferase. Slight increases in GST activity were observed in SBF animals at both 30 and
90 days (Table 6). These increases were significant only at the 2.5% dose group. Decreases in the activity were observed in the LRE groups at both 30 and 90 days, but these effects were significant only in the 8.0% group when compared with ad lib. controls.
30-Day synergism study A 30-day study was conducted following completion of the 90-day study to determine whether LRE or SBF can exert a synergistic or additive effect on induction of a single enzyme. The enzyme chosen for analysis was U D P G T based on the observation in the 90-day study that both SBF and LRE admini tered separately exerted modest increases in the activity of this enzyme. The doses administered were 0.8, 2.5 and 5% LRE, in combination with 2.5, 8 and 20% SBF. The top dose of LRE (5%) was selected based on the observed toxic effects of the compound at doses of 8 and 25% in the 90-day study. Although no adverse effects were noted for SBF, the top dose was lowered from 25% in the 90-day study to 20% to maintain a maximum of 25% dietary supplements (20% SBF + 5% LRE) in the top dose group. All mice survived until autopsy. Analysis of liver homogenates indicated a decrease in U D P G T activity in every combination dose group (Table 7). This decrease was significant at several combinations with 8 or 20% SBF but was not observed in all high SBF dose groups. The matched control diet showed a 25% increase in the enzyme activity over ad lib. controls, but this increase was not significant. DISCUSSION
Both SBF and LRE have previously been demonstrated to have anticarcinogenic properties; however, the exact mechanism of this effect is unknown. The present study was conducted to determine whether these two dietary components, either singly or in
Enzyme induction by soya bean and liquorice combination, might affect specific hepatic enzymes in B6C3FI mice that might be responsible for the chemopreventive activities of these compounds. In addition, the effects of SBF and LRE on growth, clinical signs and health of the treated animals were evaluated. Clear signs of toxicity were observed in mice maintained on LRE. Weight loss, followed by poor weight gains relative to controls, were observed throughout the 90-day study. The mice exhibited a variety of adverse clinical signs, such as depressed activity, and 30% mortality occurred at 8 and 25% LRE. Examination of internal organs revealed many mice with lesions of the kidneys and lesser adverse effects in the liver, thymus and spleen. In contrast, SBF treated mice exhibited no adverse effects. In these animals, weight gain was normal and no adverse clinical signs were observed; all mice survived to terminal autopsy, and no treatment-related lesions were observed at autopsy. Enzyme analyses led to somewhat conflicting results. Both SBF and LRE induced increased liver:body weight ratios after 30 days of treatment, but this effect was observed at 90 days only for LRE animals. This increase is supportive of enzyme induction and appears to be more pronounced with LRE treatment. However, the protein contents of S-9 fractions were not altered by either treatment, and thus a potent, generalized hypertrophy of the liver was not observed. BPH and 7-ECOD are activities of more than one isozyme of cytochrome P-450 including CYP1A1, a P-450 frequently induced in the rodent liver by foreign chemicals. Phase I enzymes may simultaneously detoxify some compounds, while metabolizing other chemicals to reactive intermediates that may bind covalently to proteins, nucleic acids or other molecules. Therefore, an increased activity of phase I enzymes does not necessarily correlate with increased chemopreventive activity. BPH showed little increase in its activity with either treatment; however, 7-ECOD exhibited the most remarkable response of any enzyme evaluated in this study. LRE significantly enhanced 7-ECOD activity at both 30 and 90 days, whereas SBF induced significant decreases at 30 days and slight, non-significant decreases at 90 days. GST and U D P G T represent two of the major enzymes involved in phase II detoxification reactions. They catalyse conjugation of reactive chemicals to glutathione and glucose, respectively, and therefore nearly always result in enhanced elimination of toxic compounds and their metabolites. Like cytochrome P-450, these enzymes also consist of several different isozymes. The substrates selected for this study (lchloro-2,4-dinitrobenzene and 4-nitrophenol for GST and UDPGT, respectively) are commonly used to provide a general indication of these enzyme activities. In the 90-day study, SBF induced modest increases in GST, but comparable decreases were
349
observed in LRE mice. Modest increases in UDPGT were observed at both 30 and 90 days for both SBF and LRE; however, these increases were significant only for the SBF groups at 90 days. UDPGT was the only enzyme for which any evidence of enhanced activity was observed for both SBF and LRE; therefore, it was selected for analysis in the 30-day synergism study. In this study, significant decreases in UDPGT activity were observed in every combination dose group relative to both the standard diet and a matched diet. Treatment groups in which U D P G T was significantly decreased also had significant increases in the liver-to-body weight. The decrease in U D P G T activity observed after combined treatment with SBF and LRE was unexpected and is difficult to explain. Many different factors including product inhibition, cross-competition of substrates and state of the membranes are known to affect dramatically UDPGT activity (Dutton and Burchell, 1977; Kasper and Henton, 1980). The complex mixtures used in the present study would be expected to have multiple effects. Another confounding variable in interpreting this study is that the increase in liver size could offset the decrease in U D P G T activity; therefore, total glucuronidation for the liver might actually be increased. One potential limitation of the design of this study was the absence of groups receiving SBF or LRE alone to reproduce the effects seen in the 90-day study. In summary, LRE induces some signs of toxicity in B6C3F~ mice at doses of 8% and above, whereas SBF does not appear to cause any adverse effects in mice at doses up to 25% in the diet when administered for up to 90 days. Both compounds produce increased liver:body weight ratios and increases in the specific activity of selected phase I or phase II enzymes. Concurrent administration of SBF and LRE does not result in enhanced U D P G T activity and, in fact, appears to decrease the activity in all combinations evaluated. Acknowledgements--The authors gratefully acknowledge the expert technical assistance of Kathy Allen and Sue LeValley in conducting enzyme assays, Sandra Phillips and Kim Haustedt in conducting animal studies, and Dr Ron Spanggord for analytical chemistry work. Dr James MacGregor provided many helpful suggestions in the design of the studies. We also thank Dr Carolyn Clifford of the National Cancer Institute and Alegria B. Caragay of Arthur D. Little for helpful suggestions in the preparation of this manuscript. This work was supported by the National Cancer Institute Contract No. NO1-CN-05267-01 under the project direction of Dr Herbert Pierson. REFERENCES
American Institute of Nutrition (1977) Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. Journal of Nutrition 107, 1340-1348 American Institute of Nutrition (1980) Second Report of the American Institute fo Nutrition Ad Hoc Committee on
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Standards for Nutritional Studies. Journal of Nutrition 110, 1726. Barnes D. S., Clapp N. K., Scott D. A., Oberst D. L. and Berry S. G. (1983) Effects of wheat, rice, corn, and soybean bran on 1,2-dimethylhydrazine-induced large bowel tumorigenesis in F344 rats. Nutrition and Cancer 5, 14. Birk Y. (1985) The Bowman-Birk inhibitor. International Journal of Peptide and Protein Research 25, 113-131. Bradford M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principal of protein dye binding. Analytical Biochemistry 72, 248-254. Bruschi S. A. and Priestly B. G. (1988) Effects of betanaphthoflavone induction on microsomal oxygenase activity and acetaminophen-mediated cytotoxicity in a differentiated human hepatoma cell line (HH1). In Vitro Toxicology 2, 91-99. Demizu S., Kajiyama K., Takahashi K., Hiraga Y., Yamamoto S., Yukiyoshi T., Okada K. and Kinoshita T. (1988) Antioxidant and antimicrobial constituents of licorice: isolation and structure elucidation of a new benzofuran derivative. Chemical and Pharmaceutical Bulletin 36, 3474-3479. DePierre J. W., Moron M. S., Johannesen K. A. M. and Ernster L. (1975) A reliable, sensitive, and convenient radioactive assay for benzpyrene monooxygenase. Analytical Biochemistry 63, 470~84. Dragani T. A., Sozzi G. and Presutti A. (1983) Comparative study of hepatic microsomal and cytosolic enzyme activities in three murine strains. Toxicology Letters 17, 215-221. Dutton G. J. and Burchell B. (1977) Newer aspects of glucuronidation. Progress in Drug Metabolism 2, 1-70. Greenlee W. F. and Poland A. (1978) An improved assay of 7-ethoxycoumarin O-deethylase activity: induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Journal of Pharmacology and Experimental Therapeutics 205, 596~05. Gumbmann M. R., Dugan G. M., Spangler W. L., Baker E. C. and Rackis J. J. (1989) Pancreatic response in rats and mice to trypsin inhibitors from soy and potato after short-and long-term dietary exposure. Journal of Nutrition 119, 1598-1609. Habig W. H., Pabst M. J. and Jakoby W. B. (1974) Glutathione S-transferases, the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130-7139. Hasdai A. and Liener I. E. (1985) The effects of soy flour and N-nitrosobis (2-oxopropyl) amine on the pancreas of the hamster. Drug-Nutrient Interactions 3, 173-179. Hasdai A, and Liener I. E. (1986) The failure of long-term feeding of raw soy four, in the presence or absence of azaserine, to induce carcinogenic changes in the mouse pancreas. Nutrition and Cancer 8, 85-91. Hatano T., Kagawa H., Yasuhara R. and Okuda T. (1988) Two new favonoids and other constituents in licorice root: their relative astringency and radical scavenging effects. Chemical and Pharmaceutical Bulletin 36, 2090-2097. Hirayama T. (1982) Relationship of soybean paste soup intake to gastric cancer risk. Nutrition and Cancer 3, 223-233. Hirayama T. (1985) A large scale cohort study on cancer risks by diet--with special reference to the risk reducing effects of green-yellow vegetable consumption. Inter-
national Symposium of the Princess Takamatsu Cancer Research Fund 16, 41-53. Iritani N., Suga A., Fukuda H., Katsurada A. and Tanaka T. (1988) Effects of dietary casein and soybean protein on triglyceride turnover in rat liver. Journal of Nutritional Science and Vitaminology 34, 309-315. Kasper, C. B. and Henton D. (1980) In Enzymatic Basis of Detoxication. Edited by W. B. Jakoby. pp. 3-41. Academic Press, New York. Leibman K. (1971) Studies on modifiers of microsomal drug oxidation. Chemico-Biological Interactions. 3, 289-290. Lucier G. W., Sonawane B. R. and McDaniel O. S. (1977) Glucuronidation and deglucuronidation reactions in hepatic and extrahepatic tissues during perinatal development. Drug Metabolism and Disposition 5, 279~84. McGuinness E. E., Morgan R. G. and Wormsley K. G. (1987) Fate of pancreatic nodules induced by raw soya flour in rats. Gut 28 (suppl.), 207-212. McGuinness E. E. and Wormsley K. G. (1986) Effects of feeding partial and intermittent raw soya flour diets on the rat pancreas. Cancer Letters 32, 73-81. Marshall M. J. and Worsfold M. (1978) Superoxide dismutase: a direct, continuous linear assay using the oxygen electrode. Analytical Biochemistry 86, 561-573. Miller N. E. and Halpert J. R. (1987) Mechanism-based inactivation of the major beta-naphthoflavone-inducible isozyme of rat liver cytochrome P-450 by the chloramphenicol analog N-(2-p-nitrophenethyl)dichloroacetamide. Drug Metabolism and Disposition 15, 846-851. Mokhtar N. M., el-Aaser A. A., el-Bolkainy M. N., Ibrahim H. A., el-Din N. B. and Moharram N. Z. (1988) Effect of soybean feeding on experimental carcinogenesis--III. Carcinogenicity of nitrite and dibutylamine in mice: a histopathological study. European Journal of Cancer and Clinical Oncology 24, 403-411. Morgan R. G., Levinson D. A., Hopwood D., Saunders J. H. and Wormsley K. G. (1977) Potentiation of the action of azaserine on the rat pancreas by raw soya bean four. Cancer Letters 3, 87-90. Naim M., Gestetner B., Zilkah S., Birk Y. and Bondi A. (1974) Soybean isoflavones. Characterization, determination, and antifungal activity. Journal of Agricultural and Food Chemistry 22, 806-810. Okada K., Tamura, Y., Yamamoto M., Inoue Y., Takagaki R., Takahashi K., Demizu S., Kajiyama K., Hiraga Y. and Kinoshita T. (1989) Identification of antimierobial and antioxidant constituents from licorice of Russian and Xinjiang origin. Chemical and Pharmaceutical Bulletin 37, 2528-2530. Tangri K. K., Seth P. K., Surendra S. P. and Bhargava K. P. (1965) Biochemical study of anti-inflammatory and anti-arthritic properties of glycyrrhetinic acid. Biochemical Pharmacology 14, 1277-1281, Wattenberg, L. W, (1985) Chemoprevention of cancer. Cancer Research 45, 1-8. Weed H. G., McGandy R. B. and Kennedy A. R. (1985) Protection against dimethylhydrazine-induced adenomatous tumors of the mouse colon by the dietary addition of an extract of soybeans containing the Bowman-Birk protease inhibitor. Carcinogenesis 6, 1239-1241. Williams, R. T. (1971) Pathways of drug metabolism. In Handbook of Experimental Pharmacology. Vol. 28. pp. 226-249. Springer-Verlag, Berlin. Witschi H. and Kennedy A. R. (1989) Modulation of lung tumor development in mice with the soybean-derived Bowman-Birk protease inhibitor. Carcinogenesis 10, 2275-2277.
0278-6915/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
EFFECTS OF SOYA BEAN FLAKES AND LIQUORICE ROOT EXTRACT ON ENZYME INDUCTION AND TOXICITY IN B6C3FI MICE J. C. MIRSALIS*,C. M. HAMILTON,J. E. SCHINDLER,C. E. GREEN and J. E. DABBS Toxicology Laboratory, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493, USA (Accepted 20 December 1992)
Abstract--Both soya bean flakes (SBF) and liquorice root extract (LRE) have previously been reported to have anticarcinogenic properties, which have been thought to be related to an increased activity of specific enzymes responsible for the detoxification of chemical carcinogens. 30- and 90-day studies were conducted in male B6C3F~ mice to determine which, if any, of several detoxification enzymes are induced by SBF or LRE. Mice fed 8 and 25% LRE showed a variety of adverse clinical signs, poor weight gain and 30% mortality. Significant increases in liver:body weight ratios were observed in both the SBF and LRE groups. No significant treatment-related gross autopsy findings were observed in any of the SBF groups. A number of abnormalities were observed in the LRE groups, including lesions of the kidney, liver, spleen and thymus. Liver samples from the 90-day study were analysed for 7-ethoxycoumarin O-deethylase (7-ECOD), benzo[a]pyrene hydroxylase (BPH), superoxide dismutase (SOD), glutathione S-transferase (GST) and UDP-glucuronyl transferase (UDPGT) at 90 days, and at an interim 30-day autopsy. No treatment-related increases were observed for BPH or SOD. Both SBF and LRE induced modest increases in UDPGT activity. SBF induced modest increases in GST activity, but LRE decreased this activity. 7-ECOD activity was significantly increased by LRE and decreased by SBF. Samples from a 30-day study in which both LRE and SBF were administered at various dose levels were examined for UDPGT activity; all dose groups showed decreases in UDPGT activity relative to controls. The results suggest that both SBF and LRE may alter the activities of specific enzymes involved in the detoxification of chemical carcinogens; however, the combination of these two foodstuffs may not produce an additive effect in B6C3F t mice.
INTRODUCTION Considerable evidence now suggests that diet plays a major role in modulating the effects of chemical carcinogens, and a wide variety of dietary components have been suggested to possess the ability to prevent the formation of tumours (Wattenberg, 1985). This 'chemopreventive' activity is observed for many naturally occurring foods, and foods that possess anticarcinogenic properties frequently share a c o m m o n attribute: they induce a variety of enzymes involved in the detoxification and excretion of carcinogenic and toxic chemicals (Williams, 1971). Understanding the effects of specific foods, separately and in combination, on enzyme induction or inhibition will provide valuable information on the mechanism of chemoprevention of specific foods. Both soya bean and liquorice have shown chemopreventive properties in a variety of models. Soya beans contain a variety of biologically active compounds; the most intensively studied have been
*To whom correspondence should be addressed. Abbreviations: BPH = benzo[a]pyrene hydroxylase; 7ECOD = 7-ethoxycoumarin O-deethylase; GCA = glycyrrhetinic acid; GCZ = glycyrrhizin; GST = glutathione S-transferase; LRE = liquorice root extract; SBF = soya bean flakes; SOD = superoxide dismutase; UDPGT = UDP-glucuronyl transferase.
the isoflavones (Naim et aL, 1974) and the protease inhibitors (Birk, 1985). Several studies have evaluated the chemopreventive effects of soya bean, but most have evaluated the effects of purified protease inhibitors. Raw soya beans or protease inhibitors have been demonstrated to be effective chemopreventive agents in a variety of animal models (Barnes et al., 1983; Mokhtar et al., 1988; Witschi and Kennedy, 1989), and limited epidemiological data also suggest a protective effect of soya bean in humans (Hirayama, 1982 and 1985). Autoclaving or chemical processing of soya beans destroys much of the protease inhibitor activity, along with the associated chemopreventive effects. Raw soya flour is capable of decreasing the pancreatic carcinogenicity of N-nitrosobis-(2-oxopropyl)amine in Syrian golden hamsters, whereas heated soya flour was ineffective in decreasing the tumour incidence (Hasdai and Liener, 1985). Protease inhibitots have been reported to cause pancreatic lesions in rats (Gumbmann et al., 1989; Hasdai and Liener, 1985; McGuinness et al., 1987; McGuinness and Wormsley, 1986; Morgan et al., 1977), but this effect does not occur in mice (Hasdai and Liener, 1986; Weed et al., 1985). Little information is available on the induction of enzymes by specific soya bean flavones, but information is available on the effects of other structurally 343
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related compounds. The flavones are classic enzyme inducers that affect a variety of phase I and phase II biotransformation pathways (Bruschi and Priestly, 1988; Dragani et al., 1983; Miller and Halpert, 1987). Hepatic lipogenic enzyme induction, fatty acid synthesis and triglyceride levels were all markedly lower in rats fed soya bean protein than in those fed casein (Iritani et al., 1988). The main biologically active component of liquorice is the triterpene glycyrrhizin (GCZ), which is a diglucuronide of glycyrrhetinic acid (GCA). In addition to GCZ and GCA, liquorice contains a variety of flavones, with the structures and content varying with the species and region of growth (Demizu et al. 1988; Hatano et al., 1988; Okada et al., 1989). GCA is a weak inhibitor of the hepatic microsomal hydroxylation of aniline and acetanilide in vitro (Leibman, 1971). GCA, methyl-GCA and GCA-diacetate prevent elevation of serum alanine aminotransferase and aspartate aminotransferase during inflammatory reactions (Tangri et al., 1965). In the present study, the effects were evaluated of processed soya bean flakes (SBF) and liquorice root extract (LRE) on the activity of several phase I and phase II detoxification enzymes in male B6C3F t mice. In addition, a variety of effects were reported of the two foodstuffs on weight gain, survival and other clinical signs. A 90-day feeding study was conducted to determine the effects of SBF and LRE administered separately. A 30-day study was then conducted in which SBF and LRE were administered in combination. The purpose of this 'synergism' study was to determine whether the two food products could produce an additive or synergistic effect on the enzyme induction observed in the 90-day study.
MATERIALS AND METHODS
Animals. Care and housing of all animals was carried out in a facility accredited by the American Association for Accreditation of Laboratory Animal Care. The 1970 Animal Welfare Act and its amendments (P.L. 89-544 and P.L. 91-579), and the principles promulgated by the National Institutes of Health (NIH Publication No. 85-23) in the Guide for the Care and Use of Laboratory Animals were adhered to at all times. Male B6C3F~ mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA, USA) and were approximately 6 wk old at the initiation of the study. Mice were individually housed in polycarbonate solid-bottomed suspended drawer-type cages containing Sani-Chips hardwood bedding (P. J. Murphy Forest Products, Montville, N J, USA). The animals were provided with deionized UV-exposed water ad lib. The animal room was maintained at 72 _+ 3°F, with a relative humidity ranging from 30 to 65% and a 12-hr light/dark cycle throughout the study. The mice were quarantined and acclimatized for 10 days before the
start of dosing. Each dose group contained five male mice in both the 90- and 30-day studies. Diets. Mice received a semi-purified diet (modified AIN-76A) consisting of 20% casein, 0.3% DL-methionine, 52% corn starch, 13% dextrose, 5% corn oil, 5% alphacel, 3.5% AIN-76 mineral premix, 1% AIN-76 vitamin premix and 0.2% choline bitartrate (American Institute of Nutrition, 1977 and 1980). All diets were in meal form and were purchased from Zeigler Bros (Gardners, PA, USA). SBF was provided by Central Soya (Fort Wayne, IN, USA; Lot RRSBM900011), and consisted of toasted flaked soya beans; this heating destroyed any protease inhibitor activity. LRE was provided by MacAndrews & Forbes Co. (Camden, NJ, USA; Lot AHLIC900004-10) and consisted of a mixture (50: 50) of powdered liquorice root from Russian and Chinese liquorice. The same batch of LRE and SBF was used for all studies. Test diets containing SBF, LRE or both, were prepared by mixing ground material into the AIN-76A diet with a twin-shelled V-blender (Patterson-Kelly Co., East Strodsburg, PA, USA). All test diets were adjusted for the loss of minerals, vitamins, choline bitartrate and DL-methionine displaced by the addition of the test compounds. Analytical chemistry studies demonstrated that the diets were stable for at least 6 wk, and homogeneity studies revealed that the coefficient of variation was less than 5 0 . For the 90-day study, animals received 0.8, 2.5, 8 or 25% SBF or LRE. For the 30-day synergism study, mice received all possible combinations of 20, 8 and 2.5% SBF, and 5, 2.5 and 0.8% LRE. Pair-fed control groups. Pair-fed control groups were added to the 90-day study; this corresponded to the make-up of the 25% SBF and LRE groups. Mice fed the highest dose of LRE showed considerable weight loss and unwillingness to eat the test diet; therefore, from wk 3 the LRE pair-fed controls were matched to the next highest dose group (8% LRE) for the remainder of the study. The pair-fed control diet consisted of AIN-76A diet with casein and dextrose added to match the protein and carbohydrate levels found in the diets containing the highest doses of SBF and LRE. The SBF pair-fed control diet consisted of 25% protein, 4.5% corn oil, 5% vitamins/minerals and 65.6% carbohydrate. The LRE pair-fed control diet consisted of 17% protein, 6.6% fat, 5% vitamins/minerals and 71.4% carbohydrate. AIN-76A diet was fed to a control group, which was allowed food ad lib. In the 30-day study, a matched diet containing the protein and carbohydrate levels of the highest SBF/LRE dose groups was prepared but was given ad lib to the mice. In-life measurements. Body weights and food co sumption were determined weekly throughout the study. Mice were observed daily throughout both studies for signs of toxicity including evaluation of appearance, posture, gait and behaviour, as well as any abnormalities in respiration and body
Enzyme induction by soya bean and liquorice
345
Table 1. Body weights and liver: body weight ratios of mice after 30 or 90 days of treatment with liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days 90 days Dose No. of Body weight No. of Body weight Treatment (%) animals (g) Liver/body animals (g) Liver/body Control 0.0 5 34.0 _+ 1.1 4.74 -+ 0.26 5 43.6 _+2.9 5.37 _+0.35 Pair-fed (SBF) SBF
0.0 0.8 2.5 8.0 25.0
Pair-fed (LRE) LRE
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
31.5 _+2.1 35. I _+2.4 33.3_+1.9 32.9 _+ 1.7 32.5_+1.7
5 31.9 _+2.1 5 32.8 + 1.5 5 28.3_+2.0 5 22.2 _+2.7* 3 24.4 __.0.4* Liver/body = 100 x liver *Lower than pair-fed control (P < 0.01; Student's t-test). "t'Greater than pair-fed control (P < 0.05; Student's t-test). :~Greater than pair-fed control (P < 0.01; Student's t-test). Values are means + SD of the numbers of mice indicated.
t e m p e r a t u r e o r u r i n e a n d faeces. All e x t e r n a l s t r u c t u r e s were e x a m i n e d a n d e a c h m o u s e w a s p a l p a t e d f o r e x t e r n a l m a s s e s . A c o m p l e t e a u t o p s y w a s perf o r m e d o n all m i c e f o l l o w i n g killing b y C O : i n h a l a tion. A complete autopsy included external e x a m i n a t i o n o f all b o d y s u r f a c e s a n d orifices, a n d o f all o r g a n s a n d t i s s u e s o f t h e a b d o m i n a l , t h o r a c i c a n d c r a n i a l cavities. L i v e r s were r e m o v e d , w e i g h e d a n d used for the evaluation of enzymes. Enzyme activity. D u r i n g a u t o p s y , t h e liver w a s e x c i s e d f r o m t h e a n i m a l a n d w e i g h e d in a c o v e r e d b e a k e r . T h e t i s s u e w a s m i n c e d , a d d e d to chilled 0.15 M-KCI b u f f e r (3 m l / g liver) a n d h o m o g e n i z e d w i t h a P o l y t r o n h o m o g e n i z e r f o r 30 sec o r less. T h e h o m o g e n a t e w a s c e n t r i f u g e d at 9 0 0 0 g f o r 15 m i n at 4°C. The supernatant containing microsomes and soluble p r o t e i n s w a s collected, a l i q u o t e d a n d s t o r e d f r o z e n a t -135°C until assayed. Each sample was measured for protein content by the spectrophotometric assay o f B r a d f o r d (1976) u s i n g C o o m a s s i e Blue dye. All b i o c h e m i c a l a s s a y v a l u e s were n o r m a l i z e d u s i n g t h e p r o t e i n c o n t e n t d a t a . Specific e n z y m e s were e v a l u a t e d
4.21 _+0.23 4.98 _+0.26~, 4.75 -+ 0.26I" 4.54 _+0.29 4.94 -+ 0.21~ 4.84 _+0.47 5.06 _+0.34 5.53_+0.67 6.60 _+0.32~ 5.82 _+0.31t weight/body weight
5 5 5 5 5
43.2 _+ 1.3 44.5 _+ 1.2 43.5_+2.2 45.3 -t- 2.4 42.0_+1.3
5.01 _+0.46 5.27 __.0.34 5.10_+0.16 5.21 ___0.21 4.90_+0.13
5 5 5 2 4
43.8 _+ 1.3 39.9 _+4.0 32.8_+2.1" 27.8 _+3.3 27.5 _+ 1,4"
4.90 _+0.27 5.22 _+0.32 6.46 -+ 0.30:~ 9.54 _+ 1.93 7.99 _+0.88~
as d e s c r i b e d below. D e e t h y l a t i o n o f 7 - e t h o x y c o u m a r i n w a s d e t e r m i n e d by m e a s u r i n g p r o d u c t formation using fluorescence spectroscopy. Tissue h o m o g e n a t e s a n d c o f a c t o r s were i n c u b a t e d as des c r i b e d b y G r e e n l e e a n d P o l a n d (1978). B e n z o [a]pyrene hydroxylase (BPH) was measured using the r a d i o a c t i v i t y a s s a y d e v e l o p e d b y D e P i e r r e et al. (1975). Glutathione S-transferase (GST) was m e a s u r e d in tissue h o m o g e n a t e s u s i n g 1-chloro-2,4d i n i t r o b e n z e n e as d e s c r i b e d b y H a b i g et aL (1974). U D P - g l u c u r o n y l t r a n s f e r a s e ( U D P G T ) activity w a s d e t e r m i n e d b y m e a s u r i n g t h e d i s a p p e a r a n c e o f 4n i t r o p h e n o l ( L u c i e r et al., 1977). S u p e r o x i d e d i s m u tase (SOD) was measured continuously with an o x y g e n electrode, w h i c h m e a s u r e s t h e rate o f o x y g e n consumption induced by the photochemical generation of superoxide from dissolved oxygen followed b y d i s m u t a t i o n to o x y g e n a n d h y d r o g e n p e r o x i d e ( M a r s h a l l a n d W o r s f o l d , 1978). Statistical a n a l y s e s were c a r r i e d o u t u s i n g S t u d e n t ' s t-test, a n d a difference at P ~< 0.05 w a s c o n s i d e r e d to be significant.
Table 2. Protein contents of mouse liver homogenates after 30 or 90 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days 90 days Dose No. of No. of Treatment (%) animals mg/ml* mg/gt animals mg/ml* mg/gt Control 0.0 5 20.2 + 1.1 60.7 + 3.3 5 20.7 + 1.7 62.0 + 5.0 Pair-fed (SBF) SBF SBF SBF SBF
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
Pair-fed (LRE) 0.0 5 LRE 0.8 5 LRE 2.5 5 LRE 8.0 2 LRE 25.0 4 *mg protein per ml S-9 (mean _+SD). tmg protein per g wet tissue (mean _+SD).
23.0_+2.1 18.7 _+2.1 20.5 _+ 1.7 19.8+2.2 20.9 _+2.6
69.1 _+6.4 56.2 _+6.4 61.4 _+5.2 59.3_+6.5 62.8 _+7.9
5 5 5 5 5
23.0_+ 1.8 19.6 _+4.0 19.5 _+0.9 17.3+ 1.0 19.9 _+ 1.4
69.1 _+5.3 58.7 _+ I 1.9 58.4 _+2.8 52.0_+3.1 59.6 _+4.3
20.0 _+2.5 22.0 _+ 1.8 23.1_+1.9 23.0 _+0.6 22.3 _+2.3
60.0 _+7.6 66.0 _+5.4 69.2_+5.8 68.9 _+ 1.9 66.8 _+6.8
5 5 5 5 3
21.5 _+2.0 20.9 _+0.9 21.8_+0.9 24.4 _+ 1.2 22.3 _+ t.5
64.5 _+5.9 62.8 _+2.7 65.4_+2.7 73.1 _+3.6 66.8 _+4.6
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Table 3. Benzo[a]pyrene hydroxylase (BPH) and superoxide dismutase (SOD) activities in the livers of mice after 30 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) BPH Dose (%)
No. of animals
Activity*
0.0
5
2.5 + 1.4
Pair-fed (SBF) SBF SBF SBF SBF
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
Pair-fed (LRE) LRE LRE LRE LRE
0.0 0.8 2.5 8.0 25.0
5 5 5 5 3
Treatment Control
SOD % Pair-fed
No. of animals
Activity*
--
5
1.24 _+ 0.65
--
2.2 + 0.9 1.8 + 0.6 2.4 + 0.8 2.5 _+ 1.0 2.3 + 0.9
100 70 109 114 105
5 5 5 5 5
0.92 + 0.79 1.43 + 0.58 1.58 + 0.39 1.29 _+0.97 0.82 +_ 0.54
100 155 172 140 89
1.7 + 1.8 ± 2.0 ± 3.1 ± 2.1 +
100 106 118 182 124
5 5 5 5 3
1.86 _+0.91 1.92 ± 1.00 0.44 + 0.53 2.12 ± 0.48 2.48 + 0.75
100 103 24t 114 133
1.2 0.9 1.6 1.2 0.4
% Pair-fed
*Enzyme activity (nmol/mg protein/min; mean _+ SD). tLower than control (P < 0.05; Student's t-test).
RESULTS
90-day study A 90-day study was conducted to determine which, if any, of several detoxification enzymes are affected by SBF or LRE administered individually in the diet. A 30-day interim autopsy was also included. The main clinical finding was poor weight gain in all mice given LRE except the lowest dose group (i.e. 0.8% LRE). Weight losses were observed in the first week and coincided with decreased food consumption during the same period. The 25% SBF group seemed to gain weight more slowly than the other SBF groups, but these differences were not significant when compared with pair-fed controls. Decreased body weights (v. pair-fed controls) were observed in the LRE groups at both 30- and 90-day autopsies (Table 1). Significant increases in liver: body weight ratios were observed in both the SBF and LRE groups at 30 days, but only at 90 days in the LRE groups. In the SBF groups, significant increases were observed at 30 days in both the absolute liver weight and liver:body weight ratio relative to pair-fed controis, but these increases were not dose related. In the
LRE groups significant increases in the absolute liver weight were not observed; however, when liver weights were adjusted for the lower increases in body weight liver-to-body weight increases were significant at the two highest doses after 30 days of treatment and at the highest and third highest doses after 90 days of treatment. The protein concentration of S-9 fractions prepared from the livers did not vary significantly from the control (Table 2). No adverse clinical effects were observed in SBF mice at either the 30- or 90-day autopsy. Animals in both the 8 and 25% LRE groups showed adverse clinical signs throughout the study including thin, hunched posture, a lump (0.5-1.0 cm) near the prepuce, alopecia on the abdomen and between the anus and the base of the tail, irritated swollen and red area between the anus and the base of the tail, rough fur, and lethargic, depressed and weak behaviour. Of 10 mice per group maintained on LRE, three in the 25% group and three in the 8% group were found dead or moribund before the scheduled autopsy. Deaths occured during wk 4 and 11 for the 25% LRE group and during wk 11 and 12 for the 8% LRE group. On the basis of the expected poor survival rate
Table 4. 7-Ethoxycoumarin O-deethylase activity in the livers of mice after 30 or 90 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days % Pair-fed
Activity*
--
5
1.33 _+ 0.64
--
0.49 0.19 0.15 0.48 0.42
100 42§ 52§ 67:~ 705
5 5 5 5 5
1.28 J- 0.23 1.22 + 0.13 1.18 ± 0.32 1.23 ± 0.38 1.08 ± 0.44
100 95 92 96 84
1.68 ± 0.60 2.45 + 0.36 2.59 _+0.52 3.49 + 0.80 4.29 ± 0.60
100 146 154 208t 255t
5 5 5 2 4
0.94 + 1.48 + 1.86 + 1.71 + 2.55 +
100 157 198t 182 271
No. of animals
Activity*
0.0
5
2.62 + 0.54
Pair-fed (SBF) SBF SBF SBF SBF
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
3.05 ± 1.29 + 1.59 + 2.03 + 2.13 ±
Pair-fed (LRE) LRE LRE LRE LRE
0.0 0.8 2.5 8.0 25.0
5 5 5 5 3
Treatment Control
90 days No. of animals
Dose (%)
*Enzyme activity (nmol/mg protein/min; mean + SD). "tGreater than control (P < 0.05; Student's t-test). ~Lower than control (P < 0.05; Student's t-test). §Lower than control (P < 0.01; Student's t-test).
0.28 0.39 0.43 0.33 1.01
% Pair-fed
Enzyme induction by soya bean and liquorice
347
Table 5. UDP-Glucuronyl transferase activity in the livers of mice after 30 or 90 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days 90 days Dose No. of No. of Treatment (%) animals Activity* % Pair-fed animals Activity* % Pair-fed Control 0.0 5 1.39 + 0.55 -5 0.63 + 0.27 -Pair-fed (SBF) 0.0 5 1.18 + 0.53 SBF 0.8 5 0.91 + 0.47 SBF 2.5 5 1.47 _+0.34 SBF 8.0 5 1.20 + 0.56 SBF 25.0 5 1.67 _+0.51 Pair-fed (LRE) 0.0 5 1.14 + 0.47 LRE 0.8 5 1.11 __.0.42 LRE 2.5 5 1.39 _+0.68 LRE 8.0 5 1.81 + 0.36 LRE 25.0 3 1.27 + 0.51 *Enzyme activity (nmol/mg protein/min; mean + SD). tGreater than control (P < 0.05; Student's t-test).
in t h e 2 5 % L R E g r o u p , f o o d c o n s u m p t i o n in t h e p a i r - f e d c o n t r o l g r o u p f o r L R E w a s m o d i f i e d to m a t c h t h a t in t h e 8 % g r o u p , r a t h e r t h a n t h a t in t h e 2 5 % g r o u p , at t h e b e g i n n i n g o f w k 3. All S B F - f e d mice survived until their scheduled autopsies. N o s i g n i f i c a n t f i n d i n g s were o b s e r v e d in a n y o f t h e S B F g r o u p s , e x c e p t a single m o u s e in t h e l o w e s t d o s e g r o u p e x h i b i t i n g a m i l d l y e n l a r g e d b l a d d e r at t h e 3 0 - d a y a u t o p s y . T h i s effect w a s n o t o b s e r v e d in t h e 9 0 - d a y s t u d y a n d is n o t c o n s i d e r e d to be t r e a t m e n t related. N o s i g n i f i c a n t f i n d i n g s were o b s e r v e d in t h e t w o l o w e s t L R E d o s e g r o u p s at 30 d a y s , b u t a n u m b e r o f a b n o r m a l i t i e s were o b s e r v e d in t h e t w o h i g h e s t L R E d o s e g r o u p s . In p a r t i c u l a r , f o u r o f five m i c e in t h e 8 % L R E g r o u p a n d o n e o f t h e t h r e e s u r v i v i n g m i c e in t h e 2 5 % g r o u p h a d a b n o r m a l k i d n e y s . O t h e r a b n o r m a l i t i e s o b s e r v e d in t h e t o p t w o LRE dose groups included small thymus, enlarged s p l e e n a n d m o t t l e d liver. T h e s e f i n d i n g s were c o n f i r m e d at t h e 9 0 - d a y a u t o p s y a n d also e x t e n d e d to i n d i v i d u a l a n i m a l s in t h e 2 . 5 % d o s e g r o u p .
Enzyme assays Benzo[a]pyrene hydroxylase. A t 30 d a y s , B P H w a s i n c r e a s e d in m i c e g i v e n 8 % L R E relative to b o t h t h e
100 77 125 102 142 100 97 122 159 111
5 5 5 5 5 5 5 5 2 4
0.87 + 0.26 1.10 + 0.27 1.25 + 0.38 1.33 + 0.23 1.45 + 0.36 0.93 + 0.27 1.14 ___0.51 0.72 +..+_0.44 1.11 + 0.43 1.10 +_0.39
100 126 144 153t 167t 100 123 77 119 118
p a i r - f e d ( 1 8 2 % ) a n d ad lib. ( 1 2 4 % ) c o n t r o l s ( T a b l e 3). B e c a u s e o f c o n s i d e r a b l e a n i m a l - t o - a n i m a l varia t i o n , this i n c r e a s e w a s n o t s i g n i f i c a n t b u t is believed to be a t r u e b i o l o g i c a l increase. N o c h a n g e s in B P H activity were i n d u c e d b y S B F at 30 d a y s . T h e e n z y m e activity w a s n o t e v a l u a t e d a f t e r 90 d a y s o f t r e a t m e n t . Superoxide dismutase. C o n s i d e r a b l e a n i m a l - t o a n i m a l v a r i a t i o n w a s o b s e r v e d in this a s s a y ( T a b l e 3). Livers from some animals had no measurable SOD activity; this lack o f r e s p o n s e w a s c o n f i r m e d for s e v e r a l s a m p l e s o n s e p a r a t e d a y s . T h e e n z y m e activity m a y h a v e b e e n lost d u e to s a m p l e p r o c e s s i n g , b u t this s e e m s u n l i k e l y since all s a m p l e s were p r o c e s s e d together. T h e r e s u l t s were h i g h l y v a r i a b l e a n d t h e r e f o r e c o n c l u s i o n s a r e difficult to d r a w . T h e 2 5 % L R E g r o u p s h o w e d a n i n c r e a s e in S O D activity o v e r p a i r - f e d ( 1 3 3 % ) a n d ad lib. ( 2 0 0 % ) c o n t r o l s ; h o w ever, t h e e n z y m e activity in L R E p a i r - f e d c o n t r o l s itself w a s h i g h e r ( 1 5 0 % ) t h a n t h a t in ad lib. c o n t r o l s . Likewise, t h e 0.8, 2.5 a n d 8 % S B F g r o u p s s h o w e d i n c r e a s e s in S O D activity o v e r p a i r - f e d c o n t r o l s . In m i c e t r e a t e d w i t h L R E , t h e o n l y s i g n i f i c a n t differe n c e w a s a d e c r e a s e in S O D activity in t h e 2 . 5 % g r o u p . I n this g r o u p , t h r e e o f five a n i m a l s h a d n o o r very low activity; this m a y n o t r e p r e s e n t a significant
Table 6. Glutathione S-transferase activity in the livers of mice after 30 or 90 days of exposure to liquorice root extract (LRE) or processed soya bean flakes (SBF) 30 days 90 days Dose No. of No. of Treatment (%) animals Activity* % Pair-fed animals Activity* % Pair-fed Control 0.0 5 101 _+ 14 -5 98 + 12 -Pair-fed (SBF) SBF SBF SBF SBF
0.0 0.8 2.5 8.0 25.0
5 5 5 5 5
90 + 12 113 + 16 117 _+9 102 _+ 13 100 + 12
Pair-fed (LRE) 0.0 5 107 __.27 LRE 0.8 5 104 + 22 LRE 2.5 5 95 __.19 LRE 8.0 5 78 + 12 LRE 25.0 3 90 __.10 *Enzyme activity (nmol/mg protein/min; mean _+SD). tGreater than control (P < 0.05; Student's t-test).
100 126 130"I" 113 I 11
5 5 5 5 5
84 +_ 15 93 + 13 96 + 10 94 + 13 99 + 16
100 III 114 112 118
100 97 89 73 84
5 5 5 2 4
100 + 90 + 87 + 91 + 95 +
100 90 87 91 95
9 10 7 2 10
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Table 7. Body weights, liver: body weight ratios and UDP-glucuronyl transferase (UDPGT) activity in the livers of mice after 30 days of exposure to liquorice root extract (LRE) and processed soya bean flakes (SBF) in combination % in diet LRE SBF None NoneS" 0.8 0.8 0.8 2.5 2.5 2.5 5.0 5.0 5.0
None None12.5 8.0 20.0 2.5 8.0 20.0 2.5 8.0 20.0
No. of animals
Body weight
Liver/body
5 5 5 5 5 5 5 5 5 5 5
23.7 + 1.1 26.0 4. 1.7:~ 26.4 4. 2.0:[: 27.2 4. 1.0~ 29.9 4. 3.4:~ 27.3+1.1§ 29.3 4. 0.8§ 28. I __. 1.4~ 23.64. 1.9 23.9 + 1.3 24.4 +_ 2.2
5.89 + 0.89 5.68 4. 0.50 7.44 _ 0.445 7.59 4. 0.53§ 5.89 4. 0.21 7.06 +- 0.52~ 7.08 4. 0.35:~ 6.68 + 0.22 7.16 __ 0.81~ 7.00 __.0.98 6.85 4. 0.80
UDPGT Activity* % Control 1.70 4. 0.66 2.13 4. 0.55 1.30 + 0.19 1.04 4. 0.36 0.79 4. 0.24 1.124.0.49 0.44 4. 0.31 0.70 + 0.34 0.96+_0.18 1.28 +_0.46 1.20 +_0.31
100 125 76 61 4611 66 2611 4111 56 75 71
*Expressed as nmol/mg protein/min. t A second control group was formulated to have equivalent levels of protein, carbohydrate and lipid as in the 5% LRE/20% SBF group and the diet was provided ad lib. ~Greater than control (P < 0.05; Student's t-test). §Greater than control (P < 0.01; Student's t-test). IILowerthan control (P < 0.05; Student's t-test). Values are means + SD of the numbers of mice indicated.
effect of LRE since a consistent dose-related effect was not observed. SOD activity was not evaluated at 90 days. 7-Ethoxycoumarin O-deethylase. No increase in 7-ECOD activity was observed for the SBF groups at 30 days; in fact, this activity was significantly lower in all SBF-fed groups than in both the SBF pair-fed and ad lib. control groups (Table 4). Slight decreases in the activity (84-96% of pairfed controls) were also observed at 90 days; however, these decreases were less pronounced and not significant. At 30 days, the LRE groups showed doserelated elevations in 7-ECOD activity over LRE pair-fed controls. At the highest dose, the activity was approximately 2.5-fold higher than in pairfed controls. The increase in 7-ECOD activity was significant at the top two doses when compared with pair-fed controls, and at the top dose only when compared with ad lib. controls. The results at 90 days were comparable with those seen at 30 days, although there were fewer mice in the two highest dose groups (only two and four surviving mice at 8 and 25% LRE, respectively); therefore, a significant difference was not observed. Dose-related increases in 7-ECOD activity (approx. two- to three-fold over pair-fed controls) were observed in all LRE-treated groups. UDP-glucuronyl transferase. Slight increases in UDPGT activity were observed at 30 days in both the 25% SBF 0 4 2 % of pair-fed controls; 121% ofadlib. controls) and 8% LRE groups (150% of pair-fed controls; 129 % of ad lib. controls), but these increases were not significant (Table 5). Increases in the enzyme activity were observed in SBF animals at 90 days and were significant at the top two doses (53 and 67% over pair-fed controls, respectively). U D P G T was selected as the single endpoint for the synergism study. Glutathione S-transferase. Slight increases in GST activity were observed in SBF animals at both 30 and
90 days (Table 6). These increases were significant only at the 2.5% dose group. Decreases in the activity were observed in the LRE groups at both 30 and 90 days, but these effects were significant only in the 8.0% group when compared with ad lib. controls.
30-Day synergism study A 30-day study was conducted following completion of the 90-day study to determine whether LRE or SBF can exert a synergistic or additive effect on induction of a single enzyme. The enzyme chosen for analysis was U D P G T based on the observation in the 90-day study that both SBF and LRE admini tered separately exerted modest increases in the activity of this enzyme. The doses administered were 0.8, 2.5 and 5% LRE, in combination with 2.5, 8 and 20% SBF. The top dose of LRE (5%) was selected based on the observed toxic effects of the compound at doses of 8 and 25% in the 90-day study. Although no adverse effects were noted for SBF, the top dose was lowered from 25% in the 90-day study to 20% to maintain a maximum of 25% dietary supplements (20% SBF + 5% LRE) in the top dose group. All mice survived until autopsy. Analysis of liver homogenates indicated a decrease in U D P G T activity in every combination dose group (Table 7). This decrease was significant at several combinations with 8 or 20% SBF but was not observed in all high SBF dose groups. The matched control diet showed a 25% increase in the enzyme activity over ad lib. controls, but this increase was not significant. DISCUSSION
Both SBF and LRE have previously been demonstrated to have anticarcinogenic properties; however, the exact mechanism of this effect is unknown. The present study was conducted to determine whether these two dietary components, either singly or in
Enzyme induction by soya bean and liquorice combination, might affect specific hepatic enzymes in B6C3FI mice that might be responsible for the chemopreventive activities of these compounds. In addition, the effects of SBF and LRE on growth, clinical signs and health of the treated animals were evaluated. Clear signs of toxicity were observed in mice maintained on LRE. Weight loss, followed by poor weight gains relative to controls, were observed throughout the 90-day study. The mice exhibited a variety of adverse clinical signs, such as depressed activity, and 30% mortality occurred at 8 and 25% LRE. Examination of internal organs revealed many mice with lesions of the kidneys and lesser adverse effects in the liver, thymus and spleen. In contrast, SBF treated mice exhibited no adverse effects. In these animals, weight gain was normal and no adverse clinical signs were observed; all mice survived to terminal autopsy, and no treatment-related lesions were observed at autopsy. Enzyme analyses led to somewhat conflicting results. Both SBF and LRE induced increased liver:body weight ratios after 30 days of treatment, but this effect was observed at 90 days only for LRE animals. This increase is supportive of enzyme induction and appears to be more pronounced with LRE treatment. However, the protein contents of S-9 fractions were not altered by either treatment, and thus a potent, generalized hypertrophy of the liver was not observed. BPH and 7-ECOD are activities of more than one isozyme of cytochrome P-450 including CYP1A1, a P-450 frequently induced in the rodent liver by foreign chemicals. Phase I enzymes may simultaneously detoxify some compounds, while metabolizing other chemicals to reactive intermediates that may bind covalently to proteins, nucleic acids or other molecules. Therefore, an increased activity of phase I enzymes does not necessarily correlate with increased chemopreventive activity. BPH showed little increase in its activity with either treatment; however, 7-ECOD exhibited the most remarkable response of any enzyme evaluated in this study. LRE significantly enhanced 7-ECOD activity at both 30 and 90 days, whereas SBF induced significant decreases at 30 days and slight, non-significant decreases at 90 days. GST and U D P G T represent two of the major enzymes involved in phase II detoxification reactions. They catalyse conjugation of reactive chemicals to glutathione and glucose, respectively, and therefore nearly always result in enhanced elimination of toxic compounds and their metabolites. Like cytochrome P-450, these enzymes also consist of several different isozymes. The substrates selected for this study (lchloro-2,4-dinitrobenzene and 4-nitrophenol for GST and UDPGT, respectively) are commonly used to provide a general indication of these enzyme activities. In the 90-day study, SBF induced modest increases in GST, but comparable decreases were
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observed in LRE mice. Modest increases in UDPGT were observed at both 30 and 90 days for both SBF and LRE; however, these increases were significant only for the SBF groups at 90 days. UDPGT was the only enzyme for which any evidence of enhanced activity was observed for both SBF and LRE; therefore, it was selected for analysis in the 30-day synergism study. In this study, significant decreases in UDPGT activity were observed in every combination dose group relative to both the standard diet and a matched diet. Treatment groups in which U D P G T was significantly decreased also had significant increases in the liver-to-body weight. The decrease in U D P G T activity observed after combined treatment with SBF and LRE was unexpected and is difficult to explain. Many different factors including product inhibition, cross-competition of substrates and state of the membranes are known to affect dramatically UDPGT activity (Dutton and Burchell, 1977; Kasper and Henton, 1980). The complex mixtures used in the present study would be expected to have multiple effects. Another confounding variable in interpreting this study is that the increase in liver size could offset the decrease in U D P G T activity; therefore, total glucuronidation for the liver might actually be increased. One potential limitation of the design of this study was the absence of groups receiving SBF or LRE alone to reproduce the effects seen in the 90-day study. In summary, LRE induces some signs of toxicity in B6C3F~ mice at doses of 8% and above, whereas SBF does not appear to cause any adverse effects in mice at doses up to 25% in the diet when administered for up to 90 days. Both compounds produce increased liver:body weight ratios and increases in the specific activity of selected phase I or phase II enzymes. Concurrent administration of SBF and LRE does not result in enhanced U D P G T activity and, in fact, appears to decrease the activity in all combinations evaluated. Acknowledgements--The authors gratefully acknowledge the expert technical assistance of Kathy Allen and Sue LeValley in conducting enzyme assays, Sandra Phillips and Kim Haustedt in conducting animal studies, and Dr Ron Spanggord for analytical chemistry work. Dr James MacGregor provided many helpful suggestions in the design of the studies. We also thank Dr Carolyn Clifford of the National Cancer Institute and Alegria B. Caragay of Arthur D. Little for helpful suggestions in the preparation of this manuscript. This work was supported by the National Cancer Institute Contract No. NO1-CN-05267-01 under the project direction of Dr Herbert Pierson. REFERENCES
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