Oct 5, 2001 - Håkan Sjunnesson, MD,1 Erik Sturegård, MD,1 Roger Willén, MD, PhD,2 and Torkel Wadström, MD, PhD1,* ...... In O. Zak, and M. E. Sande (ed.) ...
Comparative Medicine Copyright 2001 by the American Association for Laboratory Animal Science
Vol 51, No 5 October 2001 Pages 418-423
High Intake of Selenium, β-Carotene, and Vitamins A, C, and E Reduces Growth of Helicobacter pylori in the Guinea Pig Håkan Sjunnesson, MD,1 Erik Sturegård, MD,1 Roger Willén, MD, PhD,2 and Torkel Wadström, MD, PhD1,* Purpose: Helicobacter pylori is a human gastroduodenal pathogen associated with type-B gastritis and gastric cancer. Low gastric tissue antioxidant levels are believed to increase the risk of developing gastric cancer. We investigated whether dietary antioxidant levels protect against infection and type-B gastritis in H. pylori-infected guinea pigs. Methods: Dunkin-Hartley guinea pigs infected for 6 weeks with H. pylori were fed diets with various antioxidant levels. Stomach specimens were cultured, and gastritis was graded from 0 to 3. Results: Supplementation with vitamins A, C, and E and with selenium yielded H. pylori recovery from 17% of challenged animals, compared with 43% of those fed a control diet. Gastritis was scored at 0.33 and 0.93, respectively. Supplementation with only vitamin C or astaxanthin had less effect on gastritis and recovery rate. In a second experiment, gastritis score in a group given vitamins A, C, E, and selenium and β-carotene was 2.25 and in a control group, it was 2.57. The H. pylori recovery rate was 75 and 100%, respectively, with fewer colonies from animals given antioxidant supplementation (P < 0.05). Conclusions: A combination of antioxidants can protect against H. pylori infection in guinea pigs. In animal studies, antioxidant intake should be low to optimize development of H. pylori-associated disease. Furthermore we established that H. pylori causes severe gastritis in guinea pigs. The human gastric pathogen Helicobacter pylori is strongly associated with type-B gastritis, peptic ulcer disease, and gastric cancer (1-4). Some studies have indicated that differences between the prevalence of H. pylori infection and associated diseases could be partly explained by nutritional status and a regular intake of certain vitamins and antioxidants (5-8). These antioxidants can neutralize reactive oxygen metabolites (ROMs) associated with the development of chronic type-B gastritis and gastric cancer (9, 10). Increased risk of gastric cancer and precancerous conditions is associated with low plasma concentrations of α-tocopherol, vitamin C, beta carotene, and retinol, and possibly, low tissue selenium concentration (11-16). The vitamin C concentration in gastric juice is lower in patients with chronic gastritis, and in patients with H. pylori infection. In addition, concentration of vitamin C is normalized after H. pylori eradication (17-20), and vitamin C seems to inhibit H. pylori in vitro and in vivo (21). Development of animal models for H. pylori infection in recent years has made it possible to perform studies on the effect of food and intake of vitamins on H. pylori-induced gastritis and related diseases. Previous data suggested that the type of feed given to infected Balb/cA mice may influence persistence of the infection (22). Helicobacter pylori infection in guinea pigs can cause severe type-B gastritis similar to that in the normal human host (23, 24). The Mongolian gerbil is the only other small laboratory animal with comparable sensitivity in its gastric mucosal inflammatory response to H. pylori infection (25-27). Similar anatomy and physiology of the guinea pig and human stomach and the Received: 5/21/01. Revision requested: 6/27/01. Accepted: 8/24/01. 1 Department of Medical Microbiology, Dermatology and Infection, Lund University, Solveg 23, SE-223 62 Lund, Sweden and 2Department of Pathology, Sahlgrens Hospital, University of Gothenburg, Sweden. * Corresponding author.
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fact that the guinea pig is one of few animal species other than primates that cannot synthesize vitamin C makes this animal an ideal model for this type of investigation (28-31). A Helicobacter species has not yet been reported to colonize and infect the guinea pig stomach or intestines (32). In the study reported here, the effect of antioxidant intake on H. pylori infection was investigated in infected animals that were fed diets supplemented with various amounts of vitamins A, C, and E, selenium as well as selenium and astaxanthin as antioxidants (33). Astaxanthin is a carotenoid found in high concentrations in the microalga Haematococcus pluvialis (34). Astaxanthin has been found to suppress gastric inflammation as well as the bacterial load in H. pylori-infected mice (35, 36). Selenium is an important constituent of the antioxidant-associated enzyme glutathione peroxidase, which could play an important role in protecting the gastric mucosa against oxidative stress (37).
Materials and Methods Animals. Fifty-four eight-week-old male Dunkin-Hartley guinea pigs (The Sahlin farm, Malmö, Sweden), were studied in the first experiment. In a second experiment, another 20 seven- weekold male Dunkin-Hartley guinea pigs ([Mol:DH], M & B A/S, Ry, Denmark) were used. Animals of the second experiment were tested for pathogens according to the FELASA (38) standard (Table 1) and underwent a 14-day quarantine on arrival. Animals were housed in polycarbonate cages (Scanbur A/S, Køge, Denmark); three to four animals in each 590 × 385-mm cage in the first experiment and two animals in each 425 × 266-mm cage in the second experiment. Aspen chips (Tapvei Oy, Kortteinen, Finland) were used as bedding. Animals were given tap water and guinea pig chow as described later. Environmental conditions in the second experiment included > 25 conditioned fresh air changes/h,
Effect of Selenium and Anti-oxidant Vitamins on Helibacter pylori in the Guinea Pig
Table 1. Microbiological monitoring report from breeder of animals in experiment 2 No. of positive/ No. of tested
Method
Guinea pig adenovirus Lymphocytic choriomeningitis virus Pneumonia virus of mice Reovirus type 3 Sendai virus Simian virus 5
0/8 0/8 0/8 0/8 0/8 0/8
ELISA ELISA ELISA ELISA ELISA ELISA
Bacterial and fungal infections Clostridium piliforme Bordetella bronchiseptica Dermatophytes Pasteurellae Salmonellae Streptobacillus moniliformis -Hemolytic streptococci Streptococcus pneumoniae
0/8 5/10 (= positive) 0/10 0/10 0/10 0/10 0/10 0/10
ELISA Cultivation Cultivation Cultivation ELISA Cultivation Cultivation Cultivation
Viral infections
Parasitologic infections Arthropods Stereomicroscopy Helminths Eimeria spp. Giardia spp Spironucleus spp. Other flagelates Encephalitozoon cuniculi Toxoplasma gondii
Control
0/10 0/10 0/10 0/10 0/10 5/10 (=positive) 0/8 0/8
Table 2. Amounts of supplemented substances in the various diets for experiment 1
Flotation Microscopy Microscopy Microscopy Microscopy IFA IFA
IFA = Immunofluorescent antibody (assay).
temperature range of 20 to 22°C, relative humidity between 50 and 65%, and a 12-h light/dark cycle with twilight. Animals were taken care of according to the European convention for the protection of vertebrate animals used for experimental and other purposes. The animal experiments were approved by the animal experiment ethical committee, Lund University (permit No. M101-97 and M256-96). Diets. For experiment 1, four groups of animals were fed the FD1 diet from Special Diet Services (SDS), London, UK, with or without vitamin or selenium supplementation. The ingredients in the diet were: barley, wheat feed, linseed, fish meal, soya bean meal, grass meal, oat meal by-product, vitamins, and minerals. Diet 1 was the control diet, diet 2 was supplemented with vitamin C, diet 3 was supplemented with vitamins A, C, and E and with selenium, and diet 4 was supplemented with astaxanthin (Table 2). The amounts of other substances, which were constant in all four diets, were as follows: moisture, 9.5%; crude fat, 5.2%; crude protein, 19.8%; crude fiber, 11.2%; ash, 6.9%; calcium, 1.00%; phosphorus, 0.91%; sodium, 0.28%; chloride, 0.55%; potassium, 1.20%; magnesium, 0.32%; iron, 278 mg/kg; copper, 14 mg/kg; manganese, 151 mg/kg; zinc, 63 mg/kg; fluoride, 30 mg/kg; NaNO3, 910 mg/kg; NaNO2, 2.1 mg/kg; lead, 0.75 mg/kg; arsenic, 0.33 mg/kg; and cadmium, 0.09 mg/kg. Analyses of the diets were performed by Special Diet Services in the UK, prior to this study. Eighteen animals were fed diet 1, whereas 12 animals were fed diets 2, 3, and 4. Four animals fed diet 1 were kept as uninfected controls. All other animals were challenged with H. pylori. For experiment 2, the animals were allotted to three groups. One group of eight animals was fed a control diet low in antioxidant content. Eight animals of a second group were fed the same diet supplemented with additional vitamins A, C, and E, and selenium and β-carotene (Table 3). Animals of both groups were infected with H. pylori. Four animals of a third group were fed the control diet, but were not infected with H. pylori. Experimental protocol. For experiment 1, eight-week-old
Vitamin A (IU/g) Vitamin C (mg/kg) Vitamin E (mg/kg) Selenium (mg/kg) Astaxanthin (mg/kg)
4.7 300 61 0.15 0
Diet 2
Diet 3
Diet 4
4.7 3,102 61 0.15 0
55.1 3,654 232 1 0
4.7 300 61 0.15 150
Table 3. Antioxidant concentrations in diets of experiment 2 Control diet Vitamin A (IU/g) Vitamin C (mg/kg) Vitamin E (mg/kg) Selenium (mg/kg)
5.8
