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Antimicrobial Agents and Chemotherapy, December 2003, p. 3982-3984, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3982-3984.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Efficacy of Sulforaphane in Eradicating Helicobacter pylori in Human Gastric Xenografts Implanted in Nude Mice

Xavier Haristoy,1 Karine Angioi-Duprez,2 Adrien Duprez,3 and Alain Lozniewski1*

Laboratoire de Bactériologie-Virologie, UMR CNRS 75-65,1 Laboratoire de Microchirurgie Expérimentale,2 Laboratoire d'Anatomie Pathologique EMI INSERM 0014,Faculté de Médecine, Université Henri-Poincaré, Vandæuvre-les-Nancy, France3

Received 15 November 2002/ Returned for modification 24 May 2003/ Accepted 25 August 2003


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ABSTRACT
 
Sulforaphane, an isothiocyanate abundant in the form of its glucosinolate precursor in broccoli sprouts, has shown in vitro activity against Helicobacter pylori. We evaluated the effect of sulforaphane in vivo against this bacterium by using human gastric xenografts in nude mice. H. pylori was completely eradicated in 8 of the 11 sulforaphane-treated grafts. This result suggests that sulforaphane might be beneficial in the treatment of H. pylori-infected individuals.


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TEXT
 
The recommended treatment of Helicobacter pylori-associated diseases today includes a combination of two or more antibiotics with an inhibitor of acid secretion. However, even with these treatments, some bacteria may persist, in a nongrowing, tolerant form or possibly intracellularly (5, 7, 10). This may lead to eradication failure, which is defined as the persistence of H. pylori at least 1 month after the end of antimicrobial therapy (9). Moreover, development of resistance of H. pylori strains to one or more of the antibiotics commonly used has been demonstrated previously (28). Thus, a need for new therapeutic agents that are effective against extracellular and potentially intracellular forms of H. pylori exists. We recently showed that sulforaphane, an isothiocyanate abundant in the form of its glucosinolate precursor in broccoli and broccoli sprouts, is a potent bactericidal agent against both extra- and intracellular H. pylori in vitro (11). Substantial quantities of isothiocyanates (up to 100 mg daily) and even greater quantities of their glucosinolate precursors are widely consumed by humans (12, 13, 29). They may act locally within the gastrointestinal tract or may distribute systemically after conversion to their cognate isothiocyanates (12, 15, 34). In this study, we investigated the efficacy of sulforaphane in vivo against H. pylori by using a recently developed model which uses human gastric xenografts in nude mice (22).

Sulforaphane, i.e., [(-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane] was kindly provided by J. W. Fahey (Johns Hopkins University School of Medicine, Baltimore, Md.). Stock solutions were prepared in acetonitrile, and further dilutions were made in sterile water. H. pylori strain 26695, which was previously adapted for use in the gastric xenograft model (23), was used for graft inoculation. The strain was grown on Columbia agar supplemented with 10% horse blood under microaerobic conditions as previously described (22). For this isolate, the MIC of sulforaphane was 4 µg/ml, as determined by using the agar dilution method (pH 7.4) as recommended by the National Committee for Clinical Laboratory Standard (NCCLS) (30).

Xenografts exhibiting human mature gastric epithelium and acidic secretion were obtained in nude mice as previously described (22) and with the approval of the French National Consultative Ethical Committee. Bacterial inoculation was performed by using a catheter that was implanted in the xenograft lumen (22). Two weeks after inoculation, each graft was microsurgically opened. Mucus was sampled for qualitative culture onto blood agar, and three biopsies were taken from antrum-adjacent sites for quantitative culture and histology. The levels of colonization and the concentrations of intracellular bacteria were determined by quantitative culture as previously described (23). For histological studies, specimens were processed by standard methods and stained with hematoxylin-eosin to assess the intensity of gastritis and with a modified Giemsa stain for detection of H. pylori (22).

The grafted animals were then randomly divided into two groups of 11. In the first group, 0.5 ml of sterile water with 0.5% acetonitrile containing 7.5 µmol of sulforaphane (approximately 1.33 mg) was administered via catheter once a day for 5 days. The same solution without sulforaphane was administered in the same way for 5 days in the other 11 infected grafts (control group). The effect of sulforaphane on H. pylori eradication was evaluated 4 weeks after treatment per the previous recommendation for human subjects (9). At that time, the animals were euthanized and the grafts were removed and opened. Three biopsy specimens were then taken from adjacent sites in the antrum for histological examination and determination of the level of mucosal colonization as well as the concentration of intracellular bacteria concentrations as described above. Eradication was defined by the absence of H. pylori detection by both histology and culture methods. Personnel performing this evaluation were masked (blinded) with regard to the treatment status. The MIC of sulforaphane was determined for all recovered isolates obtained from each culture-positive graft.

Before treatment, all 22 inoculated grafts were equally infected by H. pylori 26695, with no significant differences in numbers of CFU between the grafts that were destined to receive sulforaphane and the controls (P = 0.34, Mann-Whitney U test) (Table 1). No organism other than H. pylori was recovered from either mucus or mucosal samples. Viable intracellular bacteria were also isolated in all the grafts and represented 0.04 to 2.5% (mean, 0.9%) of all the viable bacteria detected in the gastric mucosa. At that time, rare limited erythematous areas associated with hemorrhagic points or ulcerations were observed at the surface of the antrum in all inoculated grafts. Histological examination of antral biopsies confirmed the presence of H. pylori and showed mild inflammation and mild or moderate activity associated with diffuse interstitial edema. During the administration of sulforaphane or placebo (diluent vehicle control) and throughout the posttherapy period, no adverse reaction or change in weight or behavior was observed in the mice. Moreover, we did not observe any macroscopic or histopathological changes in internal organs at the end of the experiments. One month after the end of treatment, eradication was observed in 8 of the 11 sulforaphane-treated grafts (Table 1). Neither macroscopic abnormalities nor antral gastritis were observed in these culture-negative grafts. For the control group, there was no significant difference between the mucosal bacterial concentrations found before administration of the diluent and those found 5 weeks later (P = 0.10, Mann-Whitney U test) (Table 1). In sulforaphane-treated grafts that were not eradicated and in control grafts, both macroscopic and histological features were similar to those observed before treatment. Intracellular bacteria were detected in all control grafts (1 to 2.7% of all the viable bacteria detected) and in two of the three sulforaphane-treated grafts that were not eradicated (0.04 and 0.3% of all the viable bacteria detected). For isolates obtained from control grafts, no change in the MIC of sulforaphane was observed, whereas a fourfold increase of the MIC (to 16 µg/ml) was observed for all the isolates obtained from the three sulforaphane-treated grafts from which H. pylori was not eradicated. Such organisms were not detected after plating of the initial inoculum onto sulforaphane-containing agar.


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  		TABLE 1. Effect of sulforaphane against H. pylori 26695 in human gastric xenografts

A wide variety of plant extracts, i.e., individual plant components such as phytochemicals and phytochemical mixtures, have been shown to be active in vitro against H. pylori (1, 3, 4, 6, 8, 17-21, 24-26, 32, 33, 35, 36). A few of these phytochemicals have been tested in vivo (2, 14, 16, 20, 24, 27, 31), but clearance of H. pylori was observed only in two cases: after administration of tea catechins to H. pylori-infected gerbils (24) and after administration of goshuyu-to, a medicinal preparation which contains various plant extracts, including ginseng, to humans (16). It is therefore critical to subject all plant compounds that are active against H. pylori in vitro to in vivo studies to determine their effectiveness in whole-organism systems, since most plant-derived compounds have not proven efficacious in such testing. Thus, having recently shown that sulforaphane was active against H. pylori in vitro and that it prevented the formation of benzo[a]pyrene-induced stomach tumors in ICR mice following an estimated daily intake of 7.5 µmol for 5 weeks (11), we next evaluated its potency in eradicating H. pylori, using the same dose for 5 days, in human gastric xenografts that were implanted in nude mice. In the present work, we have shown that sulforaphane has a significant effect against H. pylori in vivo, since we observed an eradication rate of 73% for the treated group and noted no eradication in the control group. Previous studies have demonstrated that sulforaphane, which has a concentration-dependent bactericidal activity against H. pylori, may accumulate in H. pylori-infected cells and reach intracellular levels at least fivefold higher than the administered concentration, suggesting that a reason for its bactericidal activity against intracellular bacteria may be related to its accumulation in mammalian cells (11). Whether intracellular accumulation of sulforaphane occurs in vivo is not yet known, but this may be one reason that the effectiveness of sulforaphane was not influenced in our study by the presence of intracellular bacteria. A failure of complete eradication was observed in only 3 of the 11 sulforaphane-treated grafts (due to the nature of the experiment, it was impossible to know whether there was a transient effect on H. pylori colonization), and such failure was always associated with the presence of isolates for which the MIC of sulforaphane was fourfold higher than that during the initial inoculation of an isolate. This apparent reduction in susceptibility may explain, at least partially, the failure of sulforaphane to completely eradicate H. pylori in these three xenografts. Other reasons, such as the existence of subtherapeutic antibiotic concentrations or poor stability of the drug at the site of infection, cannot be excluded.

We showed that H. pylori can be eradicated from human gastric xenografts after short-term administration of sulforaphane at a dose (1.33 mg/day in each xenograft [volume, ~7 ml]; 0.19 mg/ml) that can be achieved in the human diet (100 mg/day [stomach volume, 0.5 to 1 liter]; 0.1 to 0.2 mg/liter) (12, 13, 29). Thus, the administration of sulforaphane that can be safely delivered in the diet, particularly from broccoli sprouts, could be beneficial for the treatment of H. pylori-associated gastric diseases. A sulforaphane-enriched diet might also be of value for prophylaxis against H. pylori infection and should be further evaluated. Xenografts represent the only in vivo model that permits the study of the effect of a compound on H. pylori interacting with fully differentiated human gastric mucosa. Since this model does not completely mimic the microenvironment of the infected human stomach, which is subject to the action of food, digestive physiology, and the potential coexistence of other bacterial species, further studies in humans are necessary to confirm the in vivo activity of sulforaphane against H. pylori.


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ACKNOWLEDGMENTS
 
We thank Jed W. Fahey, Katherine K. Stephenson, and Paul Talalay (Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Md.) for their gift of sulforaphane and for their expert editorial advice.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Bactériologie, Hôpital Central, 29 Avenue du Maréchal de Lattre de Tassigny, 54035 Nancy Cedex, France. Phone: (33) 3 83 85 21 96. Fax: (33) 3 83 85 26 73. E-mail: a.lozniewski{at}chu-nancy.fr. Back


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Antimicrobial Agents and Chemotherapy, December 2003, p. 3982-3984, Vol. 47, No. 12
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.12.3982-3984.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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