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Graduate Program in Nutrition and Nutrition Department, The Pennsylvania State University, University Park, PA 16802
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • garlic • processing • 7,12-dimethylbenz(a)anthracene • DNA adducts • alliinase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Conner et al. 1984
, Imai et al. 1994
,
Lawson et al. 1992
, Mathew and Augusti 1973
, Rees et al. 1993
). Evidence of garlic's
anticarcinogenic role comes from both epidemiological and laboratory
investigations (Amagase et al. 1996
, Buiatti et al. 1989
, Hussain et al. 1990
, Ip et al. 1992
, Liu et al. 1992
, Perchellet et al. 1990
, Rao et al. 1990
, Reddy et al. 1993
, Schaffer et al. 1997
, Steinmetz et al. 1994
, Sumiyoshi and Wargovich 1990
,
Wei et al. 1988
). Enhanced garlic intake was found to
correlate with a reduction in cancer risk in various parts of the
world, including China, Italy and the United States (Buiatti et al. 1989
, Steinmetz et al. 1994
, Wei et al. 1988
).
Laboratory investigations show that water-soluble and lipid-soluble
sulfur compounds in garlic provide some of its anticarcinogenic
benefits (Hussain et al. 1990
, Ip et al. 1992
, Liu et al. 1992
, Perchellet et al. 1990
, Reddy et al. 1993
, Schaffer et al. 1997
, Sumiyoshi and Wargovich 1990
). This
protection occurs in several tissues and as a result of treatment with
a variety of different types of carcinogen. While the mechanism
accounting for this protection remains unknown, changes in the
formation of carcinogens (Dion et al. 1997
,
Shenoy and Choughuley 1992
) and in bioactivation by
either phase I and II enzymes were observed (Devasagayam et al. 1982
, Ip and Lisk 1997
, Schaffer et al. 1997
, Sparnins et al. 1988
).
7,12-dimethylbenz(a)anthracene
(DMBA)6
is bioactivated to electrophilic dihydrodiolepoxides that bind to
mammary epithelial cell DNA (Digiovanni et al. 1983
).
Studies from our laboratory showed that a depression in binding as a
result of dietary garlic consumption is correlated with a reduction in
final tumor incidence (Liu et al 1992
). Thus, under
defined conditions, DMBA-induced DNA adducts can be an early biomarker
of cancer risk.
Garlic preparations vary in the amount of organosulfur compounds
(Lawson et al. 1992
). Thus, not all garlic preparations
can be assumed to be equally effective inhibitors of experimentally
induced tumors. Likewise, the method of preparation of garlic may
influence its overall capabilities to retard the cancer process.
Previous studies showed that boiling garlic in water results in a loss
of its ability to inhibit platelet aggregation (Ali 1995
, Bordia et al. 1996
). What impact of other
processing methods, including heating, has on the anticarcinogenic
potential of garlic is unclear.
The present studies were designed to address how heating, either by microwave or convection oven, influences the ability of garlic to suppress DMBA-induced mammary epithelial cell DNA adducts. These investigations were designed to test the hypothesis that heating depresses alliinase activity, which retards the formation of sulfur compounds required for the suppression in DMBA bioactivation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experiments reported herein were approved by The Pennsylvania State
University Animal Care and Use Committee and complied with Public
Health Service Guidelines for the care and use of animals for research
purposes. Female Sprague-Dawley rats (36-d-old) were purchased from
Harlan Sprague-Dawley Inc. (Indianapolis, IN) and housed separately in
screened stainless steel cages in a room with controlled temperature
(22°C) and lighting (12-h light-dark cycle). Tap water and a basal
semipurified diet (Amagase et al. 1996
) were consumed ad
libitum during all experiments. After a 5-d acclimation period, all
rats were weighed and randomly assigned to a specific treatment as
described below. In all experiments, fresh diet was provided daily.
Food intakes and body weights changes were monitored daily during each
2-wk study. In all studies, garlic preparations, commercially available
garlic constituents or carrier (corn oil or double-distilled water) was
administered by gastric gavage thrice weekly for a total of six
intubations.
Garlic used in these studies was purchased in bulk from a local
supermarket. In all studies, the quantity of garlic provided by gastric
gavage was based on a dietary intake previously shown to suppress
DMBA-induced DNA adducts (Amagase et al. 1996
). Alliin
obtained from Indofine Chemical Company (Somerville, NJ) and
S-allyl cysteine (SAC) kindly provided by Wakunaga of
America (Mission Viejo, CA) were dissolved in double-distilled water to
a final concentration of 0.105 mmol/L prior to use. Diallyl disulfide
(DADS) obtained from Fluka Chemika-Biochemika (Ronkonkoma, NY) was
dissolved in corn oil (0.105 mmol/L) prior to use.
DMBA treatment and DNA adducts determination.
DMBA obtained from Sigma Chemical Co. (St. Louis, MO) was used without
additional purification and was thoroughly dissolved in corn oil prior
to gastric gavage intubation. In all experiments, rats were 55-d-old at
the time administered DMBA (50 mg/kg body weight) by gastric gavage.
Mammary tissues were removed 24-h after DMBA treatment, rinsed in
ice-cold saline, frozen in liquid nitrogen, and stored at -80°C for
subsequent adduct analysis. Mammary epithelial cells were isolated as
previously described (Liu et al. 1992
) and suspended in
Dulbecco's modified Eagle's medium, medium 199, fetal calf serum
(Sigma Chemical) and collagenase II (Worthington Biochemical, Freehold,
NJ) with a specific activity of at least 200 units/mg dry weight. DNA
adducts isolation was accomplished by using micrococcal nuclease,
spleen phosphodiesterase, nuclease P1 and apyrase, which were purchased
from Sigma Chemical (St. Louis, MO) and detected using a
32P-postlabeling method (Schaffer et al. 1997
). Postlabeling was achieved using T4 polynucleotide kinase
obtained from U.S. Biochemical (Cleveland, OH) and
(
-32P)-ATP obtained from ICN Biomedical (Irvine, CA).
Thin layer chromatography was performed on polyethyleneimine-coated
plastic plates (Polygram CEL 300 PEI; Alltech Associates, Deerfield,
IL). Kodak XAR-OMAT film was used for autoradiography. Characterization
of individual adducts was based on the studies of Singletary et al. (1990)
.
Experimental protocols.
Garlic cloves (approximately 10 g) were peeled, chopped, suspended in corn oil and homogenized for 1 min using a Polytron to form a homogenous slurry with a final concentration of 0.7 g per 2 mL. These suspensions were kept for 24 h at 4°C prior to use. In the 2-wk experiments described below, all rats received thrice weekly gastric gavages of the carrier with or without garlic or its constituents in a 2-mL volume. In experiment 1, the ability of gastric gavage treatment with 0.7 g of garlic on DMBA-induced DNA adduct formation was examined. Rats (n = 5) were intubated with the corn oil containing or not containing a garlic suspension for 2-wk prior to DMBA treatment. Experiment 2 examined the impact of microwave heating on the anticarcinogenic potential of garlic. In this study, rats were provided either no supplemental garlic (control) or one of five different garlic (0.7 g) suspensions. The suspensions were prepared from: peeled and crushed garlic; from peeled and crushed garlic that was immediately heated in a microwave oven (600 watts; Panasonic) for either 30- or 60 s; or from a suspension from unpeeled cloves that was heated in a microwave oven for 30- or 60-s. Microwave-heated suspensions were prepared using approximately 10 g of garlic for each designated time. The mammary tissues were isolated 2-wk after experimental treatment from five rats from each of the six treatments. In experiment 3, the impact of allowing peeled garlic to "stand" for 10 min prior to microwave treatment for 60 s was evaluated. Adducts were determined from five rats per treatment. The fourth study evaluated the impact of heating using a convection oven (GE appliance Model JDP39) on garlic's ability to block DMBA bioactivation. Garlic bulbs weighing about 80 g were placed in a shallow pan with 5 mL of tap water, and heated at 176°C for 45 min and then crushed and chopped. Another garlic bulb, approximately 80 g, had its top (1.5 cm) removed by cutting and was allowed to "stand" for 10 min prior to heating at 176°C for 45 min. In study 5, rats received by gastric gavage treatment with alliin, SAC or DADS (0.105 µmol) thrice weekly for 2 wk.
Alliinase activity.
Garlic, unheated or heated by microwave, was peeled and homogenized for
1 min in 20 mmol/L of phosphate buffer (pH 7.2) containing glycerol and
pyridoxal 5'-phosphate (20 mmol/L) using a Polytron. The resulting
homogenate was centrifuged at 20,000 g for 30 min to produce
the supernatant used for assessing alliinase activity. The method of
Rabinkov et al. (1994)
was used to monitor alliinase
activity which is expressed as µmol pyruvate generated per min.
Statistical analyses.
Food intake, body weight and individual adducts were evaluated by analysis of variance and applying a Fisher's least significant difference test for mean comparisons (StatView 4.0; Abacus Concepts, Inc., Calabasas, CA). Values are means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Food intake was not influenced by the mixture provided by gastric gavage in experiments 1 through 5. Intake of rats in experiment 1 through 4 was 11.7 ± 0.3 g/d. In study 5, food intake of rats provided double-distilled water with or without alliin, or SAC was 13.9 ± 0.6, 14.2 ± 0.8, 13.9 ± 0.2 g/d, respectively, compared to 12.5 ± 0.4 g/d given DADS in corn oil (P = 0.09).
Body weight gains of rats were not influenced significantly by gastric
gavage treatment in any experiment (P 
0.05). Body
weight gains during the 2-wk experimental treatment period were 42.1,
45.1, 43.6, 43.3 and 46.5 g for studies 1–5 respectively, with a
pooled SEM of 3.6 g.
DMBA–DNA adducts.
In study 1, providing crushed garlic by gastric gavage significantly
reduced DMBA-induced DNA adduct formation by 64% (Fig. 1
).A reduction in the anti-3,4-dihydrodiol-1,2-epoxide adducts bound to
deoxyguanosine accounted for 71% of the total binding of DMBA
metabolites to mammary cell DNA. Microwave treatment of garlic for
30 s did not influence its degree of protection as evident by a 62
and 61% reduction in total adduct formation, respectively (Fig. 2
).However, microwave heating of uncrushed garlic for 60 s completely
blocked its ability to suppress adduct formation. The lack of
protection against adduct formation also occurred when the garlic was
crushed and immediately microwaved for 60 s compared to that which
was not heated (Fig. 2
). Allowing crushed garlic to stand at
room temperature for 10 min prior to heating for 60 s in a
microwave restored its anticarcinogenic properties (Fig. 3
).Microwave heating garlic for 30 s inhibited alliinase 90%
compared with unheated garlic, while microwave heating for 60 s
totally blocked the enzyme activity (Fig. 4
).
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).Cutting the top of the garlic and allowing it to stand for 10 min
before heating enabled it to retard DMBA bioactivation (Fig. 5
), although less efficiently than peeled, crushed garlic.
|
).DADS and SAC were comparable in their ability to retard DMBA-DNA adduct
formation (47 and 51%, respectively, compared with controls).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). While dietary lipids and
associated fatty acids were shown to influence the incidence of adducts
(Amagase et al. 1996
, Schaffer et al. 1997
), the present studies provide evidence that the quantity
of corn oil (2 mL) used in these intubations does not significantly
influence their overall occurrence or pattern relative to that caused
by providing water as the carrier.
Heating foods can positively or negatively influence the
availability of nutrients. Stahl and Sies (1992)
found
that lycopene bioavailability was improved by heating tomato
preparations in oil. Ali (1995)
found that boiling
garlic for 15 min prior to homogenization and extraction was
accompanied by more than a 60% reduction in its ability to inhibit
thromboxane B2 synthesis in rabbit lung. In the latter case
it is unclear how much of the diminution in efficacy related to a
leaching of the active compounds into the water medium. The present
studies suggest that heat per se, rather than leaching likely accounts
for the loss of activity. The present studies reveal that as little as
a 60-s exposure to heating by microwave, or a 45-min heating in a
convection oven, virtually eliminates the anticarcinogenic potential of
garlic against DMBA.
Heating's inactivation of garlic's protection against
DMBA-induced adduct formation may relate to a loss of alliinase
activity. Alliinase (EC 4.41.4) is a thermolabile glycoprotein
(Jansen et al. 1989
). The present studies reveal that
after heating for 30 s in a microwave oven, only about 10% of the
original alliinase activity remained. It is possible this residual
alliinase was sufficient to convert alliin to active compounds to allow
for the continuation of its ability to alter the formation of DNA
adducts. However, 60-s heating in a microwave not only resulted in
virtually undetectable alliinase activity but also eliminated any
properties involved in suppressing DMBA-induced DNA adduct formation.
It remains to be determined if the depression in alliinase activity and
the loss of the ability to alter DMBA bioactivation are directly or
indirectly associated. Evidence that biologically active compounds are
being generated following peeling of garlic is suggested by our
evidence that a "standing" time of about 10 min following crushing
amelioriates the effects of heating.
Garlic is recognized for its exceptionally high content of sulfur
compounds. Chopping or crushing of garlic releases alliinase which
rapidly converts alliin [S(+)-alkyl-L-cysteine
sulfoxide] to allicin (dialkyl thiosulfinate) (Lawson 1993
). Allicin is quite unstable and quickly is converted to
several other sulfur compounds, such as dially sulfide, DADS, ajoene,
etc. Within garlic, -glutamylcysteines serve as reserve compounds
for alliin production (Lancaster and Shaw 1989
). During
alcoholic fermentation of garlic, such as occurring in some deodorized
preparations, -glutamyl-S-allylcysteine, a precursor to
alliin, is converted to SAC (Lawson 1993
). In the
present studies, both the lipid-soluble DADS and the water-soluble SAC,
but not alliin, were effective in blocking DMBA-induced DNA adduct
formation. Although alliin was reported to have antioxidant properties
(Rabinkov et al. 1988
), others have found that, unlike
its breakdown products, it is unable to inhibit in vitro cholesterol
biosynthesis (Gebhardt 1993
). Similarly, the present
studies reveal that alliin is ineffective in retarding the
bioactivation of DMBA.
In summary, the present studies provide evidence that processing of garlic can markedly influence its effectiveness in blocking DMBA bioactivation. Additionally, these studies suggest that the generation of biologically active allyl sulfur compounds is dependent not only on total alliinase activity, but also the time that it is allowed to act. These studies point to the importance of critically examining the manner in which garlic is processed and consumed when evaluating its anticancer properties in humans.
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FOOTNOTES |
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1 These studies were supported in part by grants
from the American Institute of Cancer Research and Wakunaga of America
Co., Ltd., Mission Viejo, CA.
2 Presented in part at Experimental Biology 98
meetings, San Francisco, CA. Song, K. and Milner, J. A., Microwave
Treatment Alters Garlic's Protection Against
7,12-Dimethylbenz(a)anthracene-Induced Rat Mammary DNA Adducts. FASEB
J. 12: 4817, 1998.
3 The authors are affiliated with the Graduate
Program in Nutrition and the Nutrition Department at The Pennsylvania
State University, University Park, PA 16802.
4 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
6 Abbreviations used: Anti-dG,
anti-3,4-dihydrodiol-1,2-epoxide-deoxyguanosine; DADS, Diallyl
disulfide; DMBA, 7,12-Dimethylbenz(a)anthracene; SAC, S-allyl
cysteine.
Manuscript received August 10, 1998. Initial review completed September 9, 1998. Revision accepted December 8, 1998.
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