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Laboratoire de Nutrition et Sécurité Alimentaire, INRA, Domaine de Vilvert, 78352 Jouy en Josas, France
1To whom correspondence should be addressed.E-mail : Muriel.thomas{at}jouy.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • organosulfur compounds • garlic • iron metabolism • subtractive hybridization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Diallyl disulfide (DADS)2 is one of the most abundant molecules found in processed garlic. When a garlic clove is crushed, the odorless precursor, allin, is rapidly converted into allicin, which decomposes instantly into other oil-soluble compounds, such as diallyl sulfide (DAS) and DADS (Fig. 1A
) (2
). Among garlic constituents, DADS appears to be the most potent at reducing the growth of a wide range of cell lines (3
). In animals, DADS has been shown to decrease the number and size of chemically induced tumors (4
). The chemopreventive aspects of DADS are often correlated with its capacity to modulate activities of drug-metabolizing enzymes at different levels (mRNA, protein or enzymatic activity) (5
).
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). We therefore analyzed H- and L-ferritin mRNA sensitivity toward DADS in REL as well as in the liver of rats receiving 100 mmol/L DADS by gastric infusion. We also tested the effect of DADS on the transferrin receptor (Tfr), which delivers iron into cells (7
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DADS (CH2=CHCH2SSCH2CH=CH2; purity 80%), dimethyl sulfoxide (DMSO) and actinomycin D were purchased from Sigma-Aldrich (L’Isle d’Abeau Chesnes, France). DADS was used without further purification (8
). Two different batches of DADS were used in the study.
REL cells were isolated from newborn rat livers and cultivated as previously described (9
,10
). DADS was dissolved in 0.1% DMSO and fully homogenized with ultrasonic waves (11
). For experiments using actinomycin D, REL cells were incubated with (4 mg/L) actinomycin D for 30 min and 10 µmol/L DADS was then added to the culture medium for an additional 4 h. All treatments were repeated three times.
Subtractive method.
All procedures were performed as previously described (12
). The driver consisted of cDNA amplicons from REL cells treated for 8 h with DMSO; the tester consisted of the cells treated the same length of time with 10 µmol/L DADS. Progressive subtraction was obtained by mixing the driver and tester pools in a molar ratio increasing from 10 to 1000 and then 10,000. The low quantity of any housekeeping cDNA in the final subtractive population was a criterion for stopping the protocol (data not shown). A total of 26 fragments were sequenced; among them, 19 clones matched H-ferritin cDNA.
Rat surgery.
Wistar male rats (Iffa Credo, L’Arbresle, France) weighing 300–350 g were housed individually in metabolic cages. They were fed M25 pellets (Dietex; Saint-Gratien, France) composed of wheat, soybean meal, fish meal, dehydrated alfalfa, fine bran, molasses, and a mineral and vitamin mix. This diet contains 23% crude protein, 3% lipids, 3.3% crude fiber and 105 mg/kg iron. The surgery technique was described previously (13
). Inside the cage, rats were given free access to food and water. During the 8-d recovery period, all rats gained weight (data not shown). Thereafter, 13 mL of rapeseed oil or rapeseed oil enriched with 0.23 mL DADS (100 mmol/L) was administered through a gastric catheter connected to a peristaltic pump. Gastric administration was started at 1000 h (except for the S12 samples, which were launched at 1700 h) and continued for 4 h. Rat livers were removed immediately (S0), or at 12 (S12), 24 (S24) or 48 (S48) h after the end of the infusion under isoflurane-induced anesthesia (Abbott, Rungis, France). After gastric infusion, we verified as well as possible that the rats had not modified their food intake to any considerable degree (data not shown).
Rats administered DADS had soft feces, and in a few cases, died 
15 h after the end of infusion. In rats, lethal or toxic effects of high DADS doses have been described (14
,15
). All aspects of the protocol conformed to the International Guiding Principles for Biomedical Research Involving Animals.
Northern blot analysis.
RNA (15 µg) from cells or tissues was separated in an agarose-denaturing gel and transferred to a membrane. Blots were hybridized in Quikhyb solution (Stratagene, Amsterdam, Netherlands) with [
-32P] dCTP probes labeled by the Rediprime II system (Pharmacia Biotech, Orsay, France). The probes were obtained using the RDA procedure (H-ferritin) and by amplification from a rat liver cDNA library (L-ferritin and Tfr). All probes were verified by sequencing. A murine 18S ribosomal RNA probe was used to assess the uniformity of RNA loading.
Western blot analysis.
Total proteins were extracted from liver homogenates as previously described (16
). Protein (5 and 10 µg) was diluted in Laemmli buffer and loaded onto a 12.5% SDS-polyacrylamide gel. The weak resolution of the gel did not allow separation of the H- and L-ferritin subunits. Ferritin was revealed using peroxidase-conjugated anti-human ferritin (Rockland, Gilbertsville, Pa) (dilution 1:5000) and the enhanced chemiluminescence (ECL+) detection system (Pharmacia Biotech). The protein band was assessed according to its apparent molecular weight compared with standard proteins and to a purified human type IV ferritin (Sigma Aldrich).
Quantification and statistical analysis.
The signals on membranes were quantified using a Fla3000 imager (Fujifilm; Paris, France) and data were analyzed using Aida software (Raytes, Paris, France). Data are means ±SD. Data were analyzed by t test (Fig. 1
B) or by ANOVA and the Student-Newman-Keuls test (Fig. 2)
with SigmaStat software (L’Isle d’Abeau Chesnes, France). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The H-ferritin gene was identified by a subtractive method as a gene with heightened expression in the REL cells incubated for 8 h in the presence of 10 µmol/L DADS. The stimulation of H-ferritin mRNA by DADS treatment was confirmed by Northern blot assay (Fig. 1B
; stimulation fold: 1.9 ± 0.4, n = 4).
Both subunits of ferritin were stimulated by DADS.
Because ferritin is a multimeric protein composed of 24 subunits of H- and L-chains, we analyzed the stimulation of H- and L-ferritin mRNA after 10 µmol/L DADS treatment for 4 h (Fig. 2
). Stimulations were 1.9 ± 0.2-fold (n = 3) and 1.5 ± 0.2-fold (n = 3) induction above the basal value for H- and L-ferritin, respectively (Fig. 2
A). No further enhancement of either mRNA was found at 50 µmol/L DADS (Fig. 2
A). When REL cells were preincubated for 30 min in the presence of actinomycin D, DADS did not stimulate H- and L-ferritin mRNA (Fig. 2
B).
Stimulation of ferritin by DADS was observed in vivo.
We tested the ability of DADS to stimulate H- and L-ferritin in rats that were infused by a gastric catheter with rapeseed oil containing 100 mmol/L DADS for 4 h. Infusion of DADS increased H- and L-ferritin mRNA levels at S12, with a maximum at S24 (Fig. 3A
). At S24, H- and L-ferritin levels increased 3 ± 1.1-fold for H and 3 ± 0.14-fold for L (n = 3). Nevertheless, a slight increase in both mRNA levels occurred in rats administered rapeseed oil alone at S24 and S48, which may have been due to the oil itself. Consistent with the Northern blot analysis, the ferritin protein content was also enhanced in rats infused by DADS at S24 (Fig. 3
B). A human purified protein was loaded to control the migration profile even though it migrated slightly faster than the rat version.
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The effect of DADS on the Tfr, a protein involved in iron transport (Fig. 4
), was explored in REL cells and in rats. REL cells synthesized a high basal level of Tfr mRNA (Fig. 4
A), whereas it was virtually undetectable in rat livers at S0 (Fig. 4
B). In REL cells, the addition of 10 µmol/L DADS for 4 h did not further increase Tfr mRNA. In contrast, a 3.6 ± 0.5-fold (n = 3) Tfr mRNA stimulation occurred in DADS-treated rats at S24 (compare control and DADS-treated rats at S24) and the level remained high for 48 h. To a lesser degree Tfr mRNA increased slightly at S24 and S48 in control rats given rapeseed oil alone.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). These results suggest that DADS administration could modify iron homeostasis. Interestingly, DAS, despite its structural similarity to DADS (Fig. 1
A), does not appear to stimulate ferritin in REL cells or in vivo (data not shown, preliminary results). The stimulation of H- and L-ferritin expression was observed in REL cells at a low DADS concentration (10 µmol/L). In the gastric infusion protocol, rats were given such high doses of DADS (667 µmol/kg body weight) that the nutritional relevance of our results to humans is difficult to assess. We are currently testing lower doses of DADS in rats.
Iron’s regulation of ferritin and transferrin genes is mainly at a post-transcriptional level in opposite directions. When the intracellular iron pool is low, the translation of ferritin mRNA is impaired through an RNA/protein partnership (17
,18
), whereas the stability of Tfr mRNA is increased (19
,20
). This balance is reversed by an intracellular iron overload. The mechanism involving DADS-enhanced H- and L-ferritin mRNA seems to operate at the transcriptional level in REL cells (Fig. 2B)
. Such a transcriptional regulation has previously been described for the H- (21
,22
) as well as for the L-subunits (23
). Although DADS seems to act at the level of transcription in REL cells, it remains to be determined what the actual mechanism underlying H- and L-ferritin stimulation in the rat liver is and whether it is compatible with concomitant enhancement of Tfr mRNA. In that receptor, both transcriptional and post-transcriptional mechanisms could account for the DADS-induced stimulation, as previously described for the responsiveness of Tfr to stimulation by erythropoietin (24
). The apparent discrepancy between an enhanced Tfr mRNA and an enhanced ferritin content has already been described in regenerating rat liver triggered by carbon tetrachloride administration (25
).
To our knowledge, the effect of DADS on H- and L-ferritin and Tfr gene expression has never been described. Associations between organosulfur compounds and iron metabolism (26
,27
) are seldom suggested. Our data suggest that a high consumption of garlic could modify iron metabolism through changes in ferritin and Tfr. However, the nutritional relevance of such data to humans remains to be determined.
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FOOTNOTES |
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Manuscript received 24 May 2002. Initial review completed 10 June 2002. Revision accepted 16 September 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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