JOHN G. BABISH 2 AND GILBERT S. STOEWSAND Department of Food Science Experiment and Technology, New "fork State Agricultural Station, Cornell University, Geneva, New Yorfe 14456 ABSTRACT Leaves from a standard, insect-susceptible cauliflower variety and an insect-resistant strain were formulated at either 10 or 25% into semipurified diets for male and female weanling rats. After 3 weeks, relative liver weights, microsomal pro tein, cytochrome P-450, and activities of hepatic microsomal aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole O-demethylase, and N-methylaniline N-demethylase were determined. Growth, feed intake, and feed efficiency of male rats were not af fected by the inclusion of the dried cauliflower leaf in the diet. However, female rats exhibited a depressed feed intake and increased feed efficiency with cauliflower leaf supplemental diets. Relative liver weights increased with increasing percentage of cauliflower leaves in the diet. Hepatic microsomal enzyme response to cauliflower leaf supplementation of the diet was greater in males than in females. Only aniline hy droxylase activity remained unchanged by the test diets. Male rats showed significant increases in N- and O-demethylation with both the 10 and 25% cauliflower diets, and increased values for microsomal protein and cytochrome P-450 at the 25% supplemental level. Female rats did not show significant hepatic microsomal induction from cauliflower leaf consumption at the 10% level. However, cytochrome P-450 and the metabolism of aminopyrine and p-nitroanisole were enhanced by consumption of cauliflower leaves at 25% of their diet. None of the parameters tested in this study evidenced a difference between the two cauliflower cultivars fed to either sex. J. Nutr. JOS: 1592-1599, 1975. INDEXING KEY WORDS cauliflower •hepatic microsomal enzymes •cyto chrome P-450
Animals and man, as well as various insects, through evolutionary development have the ability to metabolically modify exogenous chemicals, e.g., drugs, food additives, contaminants, as well as nonnutrients present in natural-ingredient foods (1, 2). The enzyme systems in animais and man responsible for these biotransformations, microsomal enzymes, are found within the endoplasmic reticulum of most tissues; but hepatic microsomal enzymes have been the most extensively studied during6 the past two . decades (3). _. , , , . " c Tfl Field Observations I Rr/7eciVv7 nli>rnrpn\
( Brassica Variety 234599,
OÃ-growing inrliratp
Olerácea ) moicane
introduced "Glossy")
from is highly
This
insect
readily
Receivedfor publicationJune 9 1975
* Supported in part by Hatch Funds through the director, New York Agricultural Experiment Station. 2Tne data are Part of a tnesls submitted by John
indi
a new
Australia resistant
(P.I.to at-
for the degree of Doctor of Philosophy at Cornell ^Dickson, M. H. & Bckenrode, C. J. Variation In
ITrir-hnnluvisi (incnopiusta attacks the do-
Brassica resistance cabbage Ã-ooper and submitted Imported cnbbageworm ln thetogreenhouse and field,
^nr,l• frnrr, +Via tack Irom the /^aV>ViarY/=> cabbage Inrvnor looper
ni).3
cauliflower triât a new
mestic standard varieties of cauliflower, Natural chemicals in plants have been suggested as one basis for insect resistance or "antibiosis." However, systematic studies of this phenomenon are sparse (4). Wattenberg (5) has shown that rats fed Brussels sprouts or cabbage (both Brassica olerácea) induced the intestinal microsomal enzyme benzo[a]pyrene hydroxylase. The purpose of the present study was to determine if rats fed cauliflower leaves
G Bablsh ln partlai fulfillment of the requirements
for publication in J. Econ. Entomoi.
1592
Downloaded from https://academic.oup.com/jn/article-abstract/105/12/1592/4768703 by Tulane University Library, Serials Acquisitions Dept. user on 04 February 2019
Hepatic Microsomal Enzyme Induction in Rats Fed Varietal Cauliflower Leaves1
1593
CAULIFLOWER AND MICROSOMAL ENZYME ACTIVITY TABLE 1 Composition of diets Basal
MATERIALS
10% cauliflower*
25% cauliflower1
AND METHODS
Animals and diets. Individually caged, weanling, male and female Sprague-Dawley rats with body weights of 42-44 g were fed ad libitum one of the experimental diets listed in table 1 for 21 to 27 days. Distilled water was also available ad lib itum. Lighting in the animal quarters was set to provide 12 hours of light from 6 AM to 6 PM daily. Temperature and relative humidity were maintained at 23.5° and 55%, respectively. The variety cauliflower varie ties, i.e., the resistant ("Glossy," P.I. 234599) and the standard variety (Snowball M), were grown on experimen tal plots at the New York State Agricul tural Experiment Station without pesticide treatment. The outer leaves, before flower ing, were handpicked, trimmed of exces sive stems, washed, freeze-dried to 13.890 of their original weight, and finely ground prior to diet preparation. The Snowball M leaves already attacked by the cabbagehad looper and been exhibited an "open in terlace" appearance, whereas the "Glossy" cultivar exhibited no leaf damage. Preparation of microsomes. Starting on day 21 of the feeding period, one male and one female were chosen at random from each dietary treatment and were killed by decapitation. By this procedure, all assays were completed by day 27. In order to minimize circadian effects on microsomal enzyme activity, all animals were killed between 6 and 8 AM after an 8-hour fast. The livers were perfused with a cold 0.9% NaCl solution, weighed, and homogenized in 4 volumes of ice-cold 1.15% KC1 con taining 20 HIM Tris-HCl buffer, pH 7.4, using a Potter-EIvehjem Teflon-glass homogenizer fitted to a mechanical drill. All microsomal enzyme analyses were per formed on the 12,000 X g supernatant (postmitochondrial fraction) obtained after two successive centrifugal steps: 2,000 X g for 10 minutes and 12,000 X g for 20 minutes. Centrifugation of the postmito chondrial fraction supernatant at 105,000 X g for 60 minutes produced a pellet that was washed in 40 mM Tris-HCl buffer, pH
%
free,test)DL-MethionineCorn Casein (vitamin oilMineral mixture2Vitamin mixture3Choline (50%)SucroseCornstarchNonnutritive chloride
Downloaded from https://academic.oup.com/jn/article-abstract/105/12/1592/4768703 by Tulane University Library, Serials Acquisitions Dept. user on 04 February 2019
showed hepatic microsomal enzyme induc tion, and if there would be a further en hanced enzyme activity when fed a spe cific insect-resistant variety.
fiber4Cauliflower leaves620.00.25.05.01.00.448.415.05.0—20.00.25.05.01.00.444.411.03.010.020.00. 1Formulated with either Snowball M or Australian "Glossy" variety. ! Rogers, Q. R. & Harper, A. E. (1965) J. Nutr. 87, 267-273. "Supplies (mg/kg) in diet: thiamin-HCl, 10; riboflavin, 20; nicotinic acid, 50; calcium pantothenate, 20; pyridoxine-HC1, 4.5; folie acid, 5.0; biotin, 0.5; vitamin B-12. 0.02; menadione sodium bisulfate, 1.0; retinyl palmitate, 5,000 units/kg: ergocalciferol, 500 units/kg; di-a-tocopfceryl acetate, 75 unite/kg. 4Cellulose type, General Biochemicals, Chagrin Falls, Ohio. 6 Leaves were washed, freeze-dried, and finely chopped prior to36.7; mixing. Proximate analysis (%) "Glossy" standard cultivar:protein, fat, 5.4; fiber, 8.9; ash, 15.8. cultivar: protein, 41.2; fat, 4.4; fiber, 8.7; ash, 10.3.
7.4, and recentrifuged at 105,000 X g for 20 minutes. The washed microsomal pellet was resuspended in the 40 mM Tris-HCl buffer, pH 7.4, and used for cytochrome P-450 quantiation. All solutions used in preparing both the 12,000 X g and 105,000 X g fractions were maintained at 0-4°. Microsomal enzyme activities were deter mined within 4 hours of decapitation. Determination of microsomal enzyme ac tivity and cytochrome P-450 values. The 2-ml incubation mixture contained 1 ml of the 12,000 X g supernatant, 0.15 M KC1, 20 mM Tris-HCl buffer, pH 7.4, 1.2 mM NADP, 9.8 HIM isocitrate, 6.0 mM MgCl2, 7.5 mM semicarbazide-HCl when assaying N-demethylation, 0.18 unit of isocitrate dehydrogenase (Ls-isocitrate:NADP oxidoreductase, EC 1.1.1.42) and substrate in one of the following concentrations : aminopyrine, 7.6 mM; N-methylaniline, 7.6 mM; aniline, 8.0 mM; p-nitroanisole, 0.2 mM. This mixture was incubated under ambient air in 30-ml beakers using a Dubnoff shak ing incubator at 37°.All observations were made during a time interval when reaction rates were linear. Reactions were initiated by addition of the substrate and terminated with 1 ml of a 20% trichloroacetic acid solution. For IV-demethylation studies, aminopyrine and N-methylaniline were
1594
JOHN G. BABISH AND GILBERT S. STOEWSAND
Dietary treatment
Sex (no.)
Diet intake
Body weight
/day15.5±2.116 /rat
Basal10% variety10% standard variety25% "Glossy" variety25% standard "Glossy" varietyM
(6) (6)M F (6)F (6)M (6) (F6)M (6)F (6)M (5) F (6)g
I.iver weight wt4.00±0.21" of bodv
±32.8' 15.0Ü.3»14.8±1.0' 143.8±13.2cJ189.5±3.68±0.24'4.63±0.58»' 15.4e 12.5±0.4»lô.lil^6 135.6±16.7C193.9±23.3' 3.89±0.18"4.88±0.4212.4±1.2°15.1±2.3' 138.4±12.9C180.6±29.3'i' ±0.34"4.74±0.33»' 3.82 12.2±0.8»15.0±2.36 133.5±15.8 0.05) differences.
Female
nmole/liver 123.3±26.1« 72.2±13.8» 134.1±51.8'/ 85.7 ±27.1» 153.6 ±38.3"' 85.1 ±36.0» 179.1 ±40.2/ 106.6 ±47.7« 136.1±22.2«/ 109.1 ±10.9»
Number of animals per treatment
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and female responses to the dietary treat ments differed drastically. In males, the enhancement of enzyme activity paralleled increases observed for demethylation of aminopyrine, averaging 17 and 30% for the 10 and 25% levels of supplementation, respectively. Not only did both cauliflower varieties significantly boost N-methylaniline metabolism at the 25% dietary level, but "Glossy" significantly aggrandized demeth
1597
CAULIFLOWER AND MICROSOMAL ENZYME ACTIVITY
F-value Relative liver weight
Contrast
^-MethylAniline Aminopyrine aniline p-Nitroanisole hydroxylase JV-demethylase JV-demethylase 0-demethylase Microsomal Cytochrome activity activity activity activity protein P-450 -
nmole/mg protein/hr %ofbodywt 1. Is there an effect of dietary cauliflower leaf supplementation? (a)AtInthemales 10% level 25.39» 4.09 5.34' 10.96« At the 25% level 31.06« 3.79 34.23» 33.91» (b) In females At the 10% level 1.44 0.002 3.66 0.34 At the 25% level 14.22« 0.46 16.35« 2.01 2. Does the standard variety differ from the "Glossy"? la) In males 3.23 0.64 0.88 2.25 (b) In females 0.04 1.80 0.13 0.001 "Significant (P < 0.01).
'Significant
tng/g liver
nmole/liver
6.86* 25.43«
0.29 5.48«
1.65 4.61»
0.0007 11.54«
0.49
2.60
0.69 4.99'
2.63 0.20
0.54 0.003
1.00 0.42
(P < 0.05).
hanced by consumption of cauliflower leaves at 25% of their diet. DISCUSSION
The overall results of this study demon strate an enhancement of N- and O-demethylase enzyme activities, RLW, cyto chrome P-450, and microsomal protein related to consumption of the leaves of the cruciferous plant, Brassica olerácea. The observed increase of hepatic microsomal protein with unchanging cellular concen tration of cytochrome P-450 indicates that the response of the liver to the dietary factor(s) involved was hypertrophie rather than hyperplasic. Under such circum stances, many other cellular processes, par ticularly those related to the endoplasmic reticulum, may be affected (12, 13). In studies of the metabolism of methyl ated aminoazo dyes, Brown et al. ( 14 ) observed that the ability of mouse and rat liver homogenates to N-demethylate 3methyl-4-monomethylaminoazobenzene de pended on the nature of the diet fed. The livers of mice fed a commercial stock diet had twice the demethylation activity of those from mice fed a commercial grain or purified diet, whereas the activity of rat liver increased about 30%. Sims and Grover (15) have presented data on the metabolism of 7,12-dimethylbenz[a]-anthracene in rat liver homogenates indicat ing that rats fed a sweetened condensed milk diet showed less than 5% of the he patic enzyme activity of rats weaned to a
commercial stockproducts" diet. Other (5, 16) on "natural havestudies identified vegetable components as the source of intestinal aryl hydrocarbon hydroxylase (AHH) inducer. Loub et al. (16) provide evidence implicating naturally occurring Ã-ndolesof cruciferous plants as the factor involved in heightened intestinal AHH ac tivity. Dried powders of two cruciferous plants, cabbage and Brussels sprouts, were found to enhance intestinal O-dealkylation of phenacetin and 7-ethoxycoumarin in rats fed a 1:3 ratio of dried powder in the semisynthetic diet.6 Other dietary components in addition to vegetables contain hepatic microsomal mixed-function inducers. Earlier work in our laboratory (17) demonstrated that consumption of decaffeinated tea elevated N-demethylation of aminopyrine and 2Vmethylaniline and O-demethylation of p-nitroanisole in female rabbits. It is becom ing increasingly more evident that a myriad of minor nonnutrient constituents of plant origin have a direct influence on the microsomal mixed-function oxidases in both hepatic and nonhepatic tissues. An obvious consequence of this research is that a closer consideration of diet should be made when examining the metabolism and microsomal enzyme-inducing potential of drugs and exogenous chemicals in ani5Hsiao, K. C., Pantuck, E. J., Wattenberg, L. W., Kuntzman, R., .lacobson, M. & Conney, A. H. (1975) Stimulatory effect of a Brussels sprouts or cabbage diet on intestinal drug metabolism. Federation Proc. 34, 742. (Abstr.)
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TABLE 6 Comparing the effects of dietary cauliflower leaf supplementation and variety on liver weight, microsomal enzyme activities, microsomal protein, and cytochrome P-450 by orthogonal contrasts
1598
JOHN G. BABISH AND GILBERT S. STOEWSAND
berne (18) suggests that duplication and interpretation of experimental results could be affected when the vegetable compo nents of the diet are changed, because subtle effects can destroy the influence of the system or agent designed into the in vestigation. Aid in the interpretation of existing data on plant materials and toxins is also possible. For example, Ershoff (1921 ) has done extensive research into the modification and general reduction of toxic effects accompanying the inclusion of veg etable powders in the diet. Observed de creases in toxicity were postulated to be due exclusively to the physical properties of dietary fiber. Such a theory is probably valid in describing antitoxic effects on surface-active agents like Tween 60, but fails to account for the vegetable's protec tive effect on other nonsurface-active agents (e.g., sodium cyclamate). From present evidence it appears that enhanced metabolism of dietary toxins could be re sponsible for the observed lessening of toxic action and that this detoxication may be due to inducers present in the vegetable powders. Low levels of drug-metabolizing enzymes are found in many normal situations of rapid liver growth such as in newborn and regenerating liver, but apparently the transformation of a normal cell developing into a cancer cell is also accompanied by a decrease in the activity of several drugmetabolizing enzymes in the microsomes (3). This circumstance suggests that the depletion of these TPNH-dependent en zymes, or the smooth-surfaced endoplasmic reticulum from which they are derived, may play a role in the formation or growth of hepatomas (22). Indeed, when hydro carbons known for their ability to induce microsomal enzyme activity are admin istered at relatively low levels with chem ical carcinogens, the carcinogenic activity of the compound is nullified (23-25). Can such chemoprophylaxis be found with inducer(s) present in common vegetables? No direct data are yet available, but the emerging evidence implies that the pres ence of microsomal enzyme inducer in the diet would increase the general rate of de
toxification of noxious agents in the or ganism. ACKNOWLEDGMENTS
The authors are grateful to Dr. M. H. Dickson for the cauliflower supplied in this study and discussion on the protocol and to Miss Judy Anderson for her able technical assistance. LITERATURE CITED 1. Leopold, A. C. & Ardrey, R. (1972) Toxic substances in plants and the food habits of early man. Science 176, 512-514. 2. Parke, D. V. (1968) The Biochemistry of Foreign Compounds, Pergamon Press, Oxford. 3. Conney, A. H. (1967) Pharmacological implications of microsomal enzyme induction. Pharmacol. Rev. 19, 317-366. 4. Painter, R. H. (1968) Insect Resistance in Crop Plants. The University of Kansas Press, Lawrence. 5. Wattenberg, L. W. ( 1971 ) Studies of polycyclic hydrocarbon hydroxylases of the in testine possibly related to cancer. Cancer 28, 99-102. 6. Nash, T. (1963) The colorimetrie estima tion of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55, 416—421. 7. Imai, Y., Ito, A. & Sato, R. (1966) Evi dence for biochemically different types of vesicles in hepatic microsomal faction. J. Biochem. 60, 417-428. 8. Kato, R. & Gillette, J. R. (1965) Effect of starvation on NADPH dependent enzymes in liver microsomes of male and female rats. J. Pharmacol. Exp. Ther. 150, 279-284. 9. Schoene, B., Fleischmann, R. A., Remmer, R. & Oldershausen, H. G. (1972) Deter mination of drug metabolizing enzymes in needle biopsies of human liver. Eur. J. Clin. Pharmacol. 4, 65-73. 10. Sutherland, E. W., Cori, C. F., Haynes, R. & Olsen, N. J. (1949) Purification of the hyperglycemic-glycogenolyric factor from in sulin and gastric mucosa. J. Biol. Chem. 180, 825-837. 11. Snedecor, G. W. & Cochran, W. G. (1971) Statistical Methods, ed. 6, The Iowa State Univ. Press, Ames. 12. Schulte-Herman, R. (1974) Induction of liver growth by xenobiotic compounds and other stimuli. CRC Crit. Rev. Toxicol. 3, 97158. 13. Remmer, H., Estabrook, R. W., Schenkman, J. & Greim, H. ( 1968 ) Induction of micro somal liver enzymes. In: Enzymatic Oxida tions of Toxicants (Hodgson, E., éd.), pp. 65-88, North Carolina State Univ. Press, Raleigh. 14. Brown, R. R., Miller, J. A. & Miller, E. C. ( 1954 ) The metabolism of methylated aminoazo dyes. IV. Dietary factors enhanc ing demethylation in vitro. J. Biol. Chem. 209, 211-222.
Downloaded from https://academic.oup.com/jn/article-abstract/105/12/1592/4768703 by Tulane University Library, Serials Acquisitions Dept. user on 04 February 2019
mais and man. The variability of "natural product" diets discussed recently by New-
CAULIFLOWER AND MICROSOMAL ENZYME ACTIVITY
22.
23.
24.
25.
Protective effects of dietary fiber in rats fed toxic doses of sodium cyclamate and polyoxyethylene sorbitan monostearate (TWEEN 60). J. Food Sci. 40, 357-361. Conney, A. H. & Burns, J. J. (1963) In duced synthesis of oxidative enzymes in liver microsomes by polycyclic hydrocarbons and drugs. Advan. Enzyme Regul. 1, 189-214. Richardson, H. L., Stier, A. R. & BorsosNacht-Nebel, E. (1952) Liver tumor inhibi tion and adrenal histologie responses in rats to which 3'-methyl-4-dimethylaminoazobenzene and 20-methylcholanthrene were simultane ously administered. Cancer Res. 12, 356-361. Meechan, R., McCafferty, D. E. & Jones, R. S. (1953) 3-Methylcholanthrene as an in hibitor of hepatic cancer induced by 3-methyl4-dimethylaminoazobenzene in the diet of the rat: a determination of time relationships. Cancer Res. 13, 802-806. Miller, E. C., Miller, J. A., Brown, R. R. & MacDonald, J. C. (1958) On the pro tective action of certain polycyclic aromatic hydrocarbons against carcinogenesis by aminoazo dyes and 2-acetylaminofluorene. Cancer Res. 18, 469-477.
Downloaded from https://academic.oup.com/jn/article-abstract/105/12/1592/4768703 by Tulane University Library, Serials Acquisitions Dept. user on 04 February 2019
15. Sims, P. & Grover, P. L. (1967) Variations dependent on age and diet in the metab olism of 7,12-demethylbenz(a)anthracene by rat liver homogenates. Nature 216, 77-78. 16. Loub, W. D., Wattenberg, L. W. & Davis, D. W. ( 1975 ) Aryl hydrocarbon hydroxylase induction in rat tissues by naturally oc curring Ã-ndolesof cruciferous plants. J. Nat. Cancer Inst. 54, 985-988. 17. Babish, J. G. & Stoewsand, G. S. (1975) Effect of tea intake on induction of hepatic microsomal enzyme activity in the rabbit. Nutr. Rep. Int. 12, 109-114. 18. Newberne, P. M. (1975) Influence on pharmacological experiments of chemicals and other factors in diets of laboratory animals. Federation Proc. 34, 209-218. 19. Ershoff, B. H. (1972) Comparative effects of a purified and stock ration on sodium cyclamate toxicity in rats. Proc. Soc. Exp. Biol. Med. 141, 857-862. 20. Ershoff, B. H. & Thurston, E. W. (1974) Effects of diet on amaranth (FD & C Red no. 2) toxicity in the rat. J. Nutr. 104, 937942. 21. Ershoff, B. H. & Marshall, W. E. (1975)
1599
Animals and man, as well as various insects, through evolutionary development have the ability to metabolically modify exogenous chemicals, e.g., drugs, food additives, contaminants, as well as nonnutrients present in natural-ingredient foods (1, 2). The enzyme systems in animais and man responsible for these biotransformations, microsomal enzymes, are found within the endoplasmic reticulum of most tissues; but hepatic microsomal enzymes have been the most extensively studied during6 the past two . decades (3). _. , , , . " c Tfl Field Observations I Rr/7eciVv7 nli>rnrpn\
( Brassica Variety 234599,
OÃ-growing inrliratp
Olerácea ) moicane
introduced "Glossy")
from is highly
This
insect
readily
Receivedfor publicationJune 9 1975
* Supported in part by Hatch Funds through the director, New York Agricultural Experiment Station. 2Tne data are Part of a tnesls submitted by John
indi
a new
Australia resistant
(P.I.to at-
for the degree of Doctor of Philosophy at Cornell ^Dickson, M. H. & Bckenrode, C. J. Variation In
ITrir-hnnluvisi (incnopiusta attacks the do-
Brassica resistance cabbage Ã-ooper and submitted Imported cnbbageworm ln thetogreenhouse and field,
^nr,l• frnrr, +Via tack Irom the /^aV>ViarY/=> cabbage Inrvnor looper
ni).3
cauliflower triât a new
mestic standard varieties of cauliflower, Natural chemicals in plants have been suggested as one basis for insect resistance or "antibiosis." However, systematic studies of this phenomenon are sparse (4). Wattenberg (5) has shown that rats fed Brussels sprouts or cabbage (both Brassica olerácea) induced the intestinal microsomal enzyme benzo[a]pyrene hydroxylase. The purpose of the present study was to determine if rats fed cauliflower leaves
G Bablsh ln partlai fulfillment of the requirements
for publication in J. Econ. Entomoi.
1592
Downloaded from https://academic.oup.com/jn/article-abstract/105/12/1592/4768703 by Tulane University Library, Serials Acquisitions Dept. user on 04 February 2019
Hepatic Microsomal Enzyme Induction in Rats Fed Varietal Cauliflower Leaves1
1593
CAULIFLOWER AND MICROSOMAL ENZYME ACTIVITY TABLE 1 Composition of diets Basal
MATERIALS
10% cauliflower*
25% cauliflower1
AND METHODS
Animals and diets. Individually caged, weanling, male and female Sprague-Dawley rats with body weights of 42-44 g were fed ad libitum one of the experimental diets listed in table 1 for 21 to 27 days. Distilled water was also available ad lib itum. Lighting in the animal quarters was set to provide 12 hours of light from 6 AM to 6 PM daily. Temperature and relative humidity were maintained at 23.5° and 55%, respectively. The variety cauliflower varie ties, i.e., the resistant ("Glossy," P.I. 234599) and the standard variety (Snowball M), were grown on experimen tal plots at the New York State Agricul tural Experiment Station without pesticide treatment. The outer leaves, before flower ing, were handpicked, trimmed of exces sive stems, washed, freeze-dried to 13.890 of their original weight, and finely ground prior to diet preparation. The Snowball M leaves already attacked by the cabbagehad looper and been exhibited an "open in terlace" appearance, whereas the "Glossy" cultivar exhibited no leaf damage. Preparation of microsomes. Starting on day 21 of the feeding period, one male and one female were chosen at random from each dietary treatment and were killed by decapitation. By this procedure, all assays were completed by day 27. In order to minimize circadian effects on microsomal enzyme activity, all animals were killed between 6 and 8 AM after an 8-hour fast. The livers were perfused with a cold 0.9% NaCl solution, weighed, and homogenized in 4 volumes of ice-cold 1.15% KC1 con taining 20 HIM Tris-HCl buffer, pH 7.4, using a Potter-EIvehjem Teflon-glass homogenizer fitted to a mechanical drill. All microsomal enzyme analyses were per formed on the 12,000 X g supernatant (postmitochondrial fraction) obtained after two successive centrifugal steps: 2,000 X g for 10 minutes and 12,000 X g for 20 minutes. Centrifugation of the postmito chondrial fraction supernatant at 105,000 X g for 60 minutes produced a pellet that was washed in 40 mM Tris-HCl buffer, pH
%
free,test)DL-MethionineCorn Casein (vitamin oilMineral mixture2Vitamin mixture3Choline (50%)SucroseCornstarchNonnutritive chloride
Downloaded from https://academic.oup.com/jn/article-abstract/105/12/1592/4768703 by Tulane University Library, Serials Acquisitions Dept. user on 04 February 2019
showed hepatic microsomal enzyme induc tion, and if there would be a further en hanced enzyme activity when fed a spe cific insect-resistant variety.
fiber4Cauliflower leaves620.00.25.05.01.00.448.415.05.0—20.00.25.05.01.00.444.411.03.010.020.00. 1Formulated with either Snowball M or Australian "Glossy" variety. ! Rogers, Q. R. & Harper, A. E. (1965) J. Nutr. 87, 267-273. "Supplies (mg/kg) in diet: thiamin-HCl, 10; riboflavin, 20; nicotinic acid, 50; calcium pantothenate, 20; pyridoxine-HC1, 4.5; folie acid, 5.0; biotin, 0.5; vitamin B-12. 0.02; menadione sodium bisulfate, 1.0; retinyl palmitate, 5,000 units/kg: ergocalciferol, 500 units/kg; di-a-tocopfceryl acetate, 75 unite/kg. 4Cellulose type, General Biochemicals, Chagrin Falls, Ohio. 6 Leaves were washed, freeze-dried, and finely chopped prior to36.7; mixing. Proximate analysis (%) "Glossy" standard cultivar:protein, fat, 5.4; fiber, 8.9; ash, 15.8. cultivar: protein, 41.2; fat, 4.4; fiber, 8.7; ash, 10.3.
7.4, and recentrifuged at 105,000 X g for 20 minutes. The washed microsomal pellet was resuspended in the 40 mM Tris-HCl buffer, pH 7.4, and used for cytochrome P-450 quantiation. All solutions used in preparing both the 12,000 X g and 105,000 X g fractions were maintained at 0-4°. Microsomal enzyme activities were deter mined within 4 hours of decapitation. Determination of microsomal enzyme ac tivity and cytochrome P-450 values. The 2-ml incubation mixture contained 1 ml of the 12,000 X g supernatant, 0.15 M KC1, 20 mM Tris-HCl buffer, pH 7.4, 1.2 mM NADP, 9.8 HIM isocitrate, 6.0 mM MgCl2, 7.5 mM semicarbazide-HCl when assaying N-demethylation, 0.18 unit of isocitrate dehydrogenase (Ls-isocitrate:NADP oxidoreductase, EC 1.1.1.42) and substrate in one of the following concentrations : aminopyrine, 7.6 mM; N-methylaniline, 7.6 mM; aniline, 8.0 mM; p-nitroanisole, 0.2 mM. This mixture was incubated under ambient air in 30-ml beakers using a Dubnoff shak ing incubator at 37°.All observations were made during a time interval when reaction rates were linear. Reactions were initiated by addition of the substrate and terminated with 1 ml of a 20% trichloroacetic acid solution. For IV-demethylation studies, aminopyrine and N-methylaniline were
1594
JOHN G. BABISH AND GILBERT S. STOEWSAND
Dietary treatment
Sex (no.)
Diet intake
Body weight
/day15.5±2.116 /rat
Basal10% variety10% standard variety25% "Glossy" variety25% standard "Glossy" varietyM
(6) (6)M F (6)F (6)M (6) (F6)M (6)F (6)M (5) F (6)g
I.iver weight wt4.00±0.21" of bodv
±32.8' 15.0Ü.3»14.8±1.0' 143.8±13.2cJ189.5±3.68±0.24'4.63±0.58»' 15.4e 12.5±0.4»lô.lil^6 135.6±16.7C193.9±23.3' 3.89±0.18"4.88±0.4212.4±1.2°15.1±2.3' 138.4±12.9C180.6±29.3'i' ±0.34"4.74±0.33»' 3.82 12.2±0.8»15.0±2.36 133.5±15.8 0.05) differences.
Female
nmole/liver 123.3±26.1« 72.2±13.8» 134.1±51.8'/ 85.7 ±27.1» 153.6 ±38.3"' 85.1 ±36.0» 179.1 ±40.2/ 106.6 ±47.7« 136.1±22.2«/ 109.1 ±10.9»
Number of animals per treatment
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and female responses to the dietary treat ments differed drastically. In males, the enhancement of enzyme activity paralleled increases observed for demethylation of aminopyrine, averaging 17 and 30% for the 10 and 25% levels of supplementation, respectively. Not only did both cauliflower varieties significantly boost N-methylaniline metabolism at the 25% dietary level, but "Glossy" significantly aggrandized demeth
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CAULIFLOWER AND MICROSOMAL ENZYME ACTIVITY
F-value Relative liver weight
Contrast
^-MethylAniline Aminopyrine aniline p-Nitroanisole hydroxylase JV-demethylase JV-demethylase 0-demethylase Microsomal Cytochrome activity activity activity activity protein P-450 -
nmole/mg protein/hr %ofbodywt 1. Is there an effect of dietary cauliflower leaf supplementation? (a)AtInthemales 10% level 25.39» 4.09 5.34' 10.96« At the 25% level 31.06« 3.79 34.23» 33.91» (b) In females At the 10% level 1.44 0.002 3.66 0.34 At the 25% level 14.22« 0.46 16.35« 2.01 2. Does the standard variety differ from the "Glossy"? la) In males 3.23 0.64 0.88 2.25 (b) In females 0.04 1.80 0.13 0.001 "Significant (P < 0.01).
'Significant
tng/g liver
nmole/liver
6.86* 25.43«
0.29 5.48«
1.65 4.61»
0.0007 11.54«
0.49
2.60
0.69 4.99'
2.63 0.20
0.54 0.003
1.00 0.42
(P < 0.05).
hanced by consumption of cauliflower leaves at 25% of their diet. DISCUSSION
The overall results of this study demon strate an enhancement of N- and O-demethylase enzyme activities, RLW, cyto chrome P-450, and microsomal protein related to consumption of the leaves of the cruciferous plant, Brassica olerácea. The observed increase of hepatic microsomal protein with unchanging cellular concen tration of cytochrome P-450 indicates that the response of the liver to the dietary factor(s) involved was hypertrophie rather than hyperplasic. Under such circum stances, many other cellular processes, par ticularly those related to the endoplasmic reticulum, may be affected (12, 13). In studies of the metabolism of methyl ated aminoazo dyes, Brown et al. ( 14 ) observed that the ability of mouse and rat liver homogenates to N-demethylate 3methyl-4-monomethylaminoazobenzene de pended on the nature of the diet fed. The livers of mice fed a commercial stock diet had twice the demethylation activity of those from mice fed a commercial grain or purified diet, whereas the activity of rat liver increased about 30%. Sims and Grover (15) have presented data on the metabolism of 7,12-dimethylbenz[a]-anthracene in rat liver homogenates indicat ing that rats fed a sweetened condensed milk diet showed less than 5% of the he patic enzyme activity of rats weaned to a
commercial stockproducts" diet. Other (5, 16) on "natural havestudies identified vegetable components as the source of intestinal aryl hydrocarbon hydroxylase (AHH) inducer. Loub et al. (16) provide evidence implicating naturally occurring Ã-ndolesof cruciferous plants as the factor involved in heightened intestinal AHH ac tivity. Dried powders of two cruciferous plants, cabbage and Brussels sprouts, were found to enhance intestinal O-dealkylation of phenacetin and 7-ethoxycoumarin in rats fed a 1:3 ratio of dried powder in the semisynthetic diet.6 Other dietary components in addition to vegetables contain hepatic microsomal mixed-function inducers. Earlier work in our laboratory (17) demonstrated that consumption of decaffeinated tea elevated N-demethylation of aminopyrine and 2Vmethylaniline and O-demethylation of p-nitroanisole in female rabbits. It is becom ing increasingly more evident that a myriad of minor nonnutrient constituents of plant origin have a direct influence on the microsomal mixed-function oxidases in both hepatic and nonhepatic tissues. An obvious consequence of this research is that a closer consideration of diet should be made when examining the metabolism and microsomal enzyme-inducing potential of drugs and exogenous chemicals in ani5Hsiao, K. C., Pantuck, E. J., Wattenberg, L. W., Kuntzman, R., .lacobson, M. & Conney, A. H. (1975) Stimulatory effect of a Brussels sprouts or cabbage diet on intestinal drug metabolism. Federation Proc. 34, 742. (Abstr.)
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TABLE 6 Comparing the effects of dietary cauliflower leaf supplementation and variety on liver weight, microsomal enzyme activities, microsomal protein, and cytochrome P-450 by orthogonal contrasts
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JOHN G. BABISH AND GILBERT S. STOEWSAND
berne (18) suggests that duplication and interpretation of experimental results could be affected when the vegetable compo nents of the diet are changed, because subtle effects can destroy the influence of the system or agent designed into the in vestigation. Aid in the interpretation of existing data on plant materials and toxins is also possible. For example, Ershoff (1921 ) has done extensive research into the modification and general reduction of toxic effects accompanying the inclusion of veg etable powders in the diet. Observed de creases in toxicity were postulated to be due exclusively to the physical properties of dietary fiber. Such a theory is probably valid in describing antitoxic effects on surface-active agents like Tween 60, but fails to account for the vegetable's protec tive effect on other nonsurface-active agents (e.g., sodium cyclamate). From present evidence it appears that enhanced metabolism of dietary toxins could be re sponsible for the observed lessening of toxic action and that this detoxication may be due to inducers present in the vegetable powders. Low levels of drug-metabolizing enzymes are found in many normal situations of rapid liver growth such as in newborn and regenerating liver, but apparently the transformation of a normal cell developing into a cancer cell is also accompanied by a decrease in the activity of several drugmetabolizing enzymes in the microsomes (3). This circumstance suggests that the depletion of these TPNH-dependent en zymes, or the smooth-surfaced endoplasmic reticulum from which they are derived, may play a role in the formation or growth of hepatomas (22). Indeed, when hydro carbons known for their ability to induce microsomal enzyme activity are admin istered at relatively low levels with chem ical carcinogens, the carcinogenic activity of the compound is nullified (23-25). Can such chemoprophylaxis be found with inducer(s) present in common vegetables? No direct data are yet available, but the emerging evidence implies that the pres ence of microsomal enzyme inducer in the diet would increase the general rate of de
toxification of noxious agents in the or ganism. ACKNOWLEDGMENTS
The authors are grateful to Dr. M. H. Dickson for the cauliflower supplied in this study and discussion on the protocol and to Miss Judy Anderson for her able technical assistance. LITERATURE CITED 1. Leopold, A. C. & Ardrey, R. (1972) Toxic substances in plants and the food habits of early man. Science 176, 512-514. 2. Parke, D. V. (1968) The Biochemistry of Foreign Compounds, Pergamon Press, Oxford. 3. Conney, A. H. (1967) Pharmacological implications of microsomal enzyme induction. Pharmacol. Rev. 19, 317-366. 4. Painter, R. H. (1968) Insect Resistance in Crop Plants. The University of Kansas Press, Lawrence. 5. Wattenberg, L. W. ( 1971 ) Studies of polycyclic hydrocarbon hydroxylases of the in testine possibly related to cancer. Cancer 28, 99-102. 6. Nash, T. (1963) The colorimetrie estima tion of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55, 416—421. 7. Imai, Y., Ito, A. & Sato, R. (1966) Evi dence for biochemically different types of vesicles in hepatic microsomal faction. J. Biochem. 60, 417-428. 8. Kato, R. & Gillette, J. R. (1965) Effect of starvation on NADPH dependent enzymes in liver microsomes of male and female rats. J. Pharmacol. Exp. Ther. 150, 279-284. 9. Schoene, B., Fleischmann, R. A., Remmer, R. & Oldershausen, H. G. (1972) Deter mination of drug metabolizing enzymes in needle biopsies of human liver. Eur. J. Clin. Pharmacol. 4, 65-73. 10. Sutherland, E. W., Cori, C. F., Haynes, R. & Olsen, N. J. (1949) Purification of the hyperglycemic-glycogenolyric factor from in sulin and gastric mucosa. J. Biol. Chem. 180, 825-837. 11. Snedecor, G. W. & Cochran, W. G. (1971) Statistical Methods, ed. 6, The Iowa State Univ. Press, Ames. 12. Schulte-Herman, R. (1974) Induction of liver growth by xenobiotic compounds and other stimuli. CRC Crit. Rev. Toxicol. 3, 97158. 13. Remmer, H., Estabrook, R. W., Schenkman, J. & Greim, H. ( 1968 ) Induction of micro somal liver enzymes. In: Enzymatic Oxida tions of Toxicants (Hodgson, E., éd.), pp. 65-88, North Carolina State Univ. Press, Raleigh. 14. Brown, R. R., Miller, J. A. & Miller, E. C. ( 1954 ) The metabolism of methylated aminoazo dyes. IV. Dietary factors enhanc ing demethylation in vitro. J. Biol. Chem. 209, 211-222.
Downloaded from https://academic.oup.com/jn/article-abstract/105/12/1592/4768703 by Tulane University Library, Serials Acquisitions Dept. user on 04 February 2019
mais and man. The variability of "natural product" diets discussed recently by New-
CAULIFLOWER AND MICROSOMAL ENZYME ACTIVITY
22.
23.
24.
25.
Protective effects of dietary fiber in rats fed toxic doses of sodium cyclamate and polyoxyethylene sorbitan monostearate (TWEEN 60). J. Food Sci. 40, 357-361. Conney, A. H. & Burns, J. J. (1963) In duced synthesis of oxidative enzymes in liver microsomes by polycyclic hydrocarbons and drugs. Advan. Enzyme Regul. 1, 189-214. Richardson, H. L., Stier, A. R. & BorsosNacht-Nebel, E. (1952) Liver tumor inhibi tion and adrenal histologie responses in rats to which 3'-methyl-4-dimethylaminoazobenzene and 20-methylcholanthrene were simultane ously administered. Cancer Res. 12, 356-361. Meechan, R., McCafferty, D. E. & Jones, R. S. (1953) 3-Methylcholanthrene as an in hibitor of hepatic cancer induced by 3-methyl4-dimethylaminoazobenzene in the diet of the rat: a determination of time relationships. Cancer Res. 13, 802-806. Miller, E. C., Miller, J. A., Brown, R. R. & MacDonald, J. C. (1958) On the pro tective action of certain polycyclic aromatic hydrocarbons against carcinogenesis by aminoazo dyes and 2-acetylaminofluorene. Cancer Res. 18, 469-477.
Downloaded from https://academic.oup.com/jn/article-abstract/105/12/1592/4768703 by Tulane University Library, Serials Acquisitions Dept. user on 04 February 2019
15. Sims, P. & Grover, P. L. (1967) Variations dependent on age and diet in the metab olism of 7,12-demethylbenz(a)anthracene by rat liver homogenates. Nature 216, 77-78. 16. Loub, W. D., Wattenberg, L. W. & Davis, D. W. ( 1975 ) Aryl hydrocarbon hydroxylase induction in rat tissues by naturally oc curring Ã-ndolesof cruciferous plants. J. Nat. Cancer Inst. 54, 985-988. 17. Babish, J. G. & Stoewsand, G. S. (1975) Effect of tea intake on induction of hepatic microsomal enzyme activity in the rabbit. Nutr. Rep. Int. 12, 109-114. 18. Newberne, P. M. (1975) Influence on pharmacological experiments of chemicals and other factors in diets of laboratory animals. Federation Proc. 34, 209-218. 19. Ershoff, B. H. (1972) Comparative effects of a purified and stock ration on sodium cyclamate toxicity in rats. Proc. Soc. Exp. Biol. Med. 141, 857-862. 20. Ershoff, B. H. & Thurston, E. W. (1974) Effects of diet on amaranth (FD & C Red no. 2) toxicity in the rat. J. Nutr. 104, 937942. 21. Ershoff, B. H. & Marshall, W. E. (1975)
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