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Applied and Environmental Microbiology, November 2006, p. 7398-7400, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01348-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Lactobacillus rhamnosus Strain GG Modulates Intestinal Absorption, Fecal Excretion, and Toxicity of Aflatoxin B₁ in Rats

S. Gratz,^1,2* M. Täubel,² R. O. Juvonen,³ M. Viluksela,⁴ P. C. Turner,⁵ H. Mykkänen,¹ and H. El-Nezami²

Department of Clinical Nutrition, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland,¹ Food and Health Research Centre, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland,² Department of Pharmacology and Toxicology, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland,³ National Public Health Institute, Laboratory of Toxicology, P.O. Box 95, 70701 Kuopio, Finland,⁴ Centre for Epidemiology and Biostatistics, Molecular Epidemiology Unit, LIGHT Laboratories, University of Leeds, LS2 9JT Leeds, United Kingdom⁵

Received 12 June 2006/ Accepted 4 September 2006

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In this study, the modulation of aflatoxin B₁ (AFB₁) uptake in rats by administration of the probiotic Lactobacillus rhamnosus GG was demonstrated. Fecal AFB₁ excretion in GG-treated rats was increased via bacterial AFB₁ binding. Furthermore, AFB₁-associated growth faltering and liver injury were alleviated with GG treatment.

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Aflatoxins are toxic and carcinogenic fungal metabolites that frequently contaminate staple crops (1). Interventions in aflatoxin exposure focus either on improving crop quality and storage (20) or on altering aflatoxin bioavailability on the individual level, using various adsorbents (5, 18). However, no safe means are presently available to completely protect humans from aflatoxin exposure. Our previous work with lactic acid bacteria revealed that the probiotic strain Lactobacillus rhamnosus strain GG (ATCC 53013) efficiently binds several mycotoxins, including aflatoxin B₁ (AFB₁) and aflatoxin M₁ (AFM₁), its hydroxylated metabolite, in vitro (6, 7, 9, 10, 19). Even though heat-killed bacteria have the highest binding capacities, most studies use viable bacteria, which are more relevant in probiotic products intended for human consumption. Acid, intestinal enzymes, and intestinal mucus were shown not to interfere with AFB₁ binding of this probiotic (6, 7, 11, 12). GG was subsequently demonstrated to bind AFB₁ and to reduce its uptake into intestinal tissue in the ligated duodenal loops of 1-week-old chicks (8). However, this ex vivo method has its limitations in simulating intestinal conditions, and we therefore conducted an in vivo single-dose experiment in rats, investigating the effects of GG on AFB₁ absorption and toxic effects. Five-week-old Han-Wistar rats were kept individually in metabolic cages on standard powdered feed and water ad libitum. After acclimatization, the rats (n = 12/group) received either vehicle (phosphate-buffered saline [PBS]) or probiotic suspension (5 x 10¹⁰ CFU GG/0.5 ml PBS, prepared by directly suspending lyophilized bacteria in PBS) by oral gavage daily for 3 days before and 3 days after a single oral dose of AFB₁ (1.5 mg or 4.8 µmol/kg of body weight in dimethyl sulfoxide [Sigma-Aldrich, St. Louis, Mo.]). Four additional rats served as untreated controls. Body weight was recorded at the beginning of the study (prior to GG treatment), on the day of AFB₁ dosing, and at the end of the study. Urine and fecal samples were collected daily, weighed, and stored at –20°C. At the end of the study, blood samples were taken by cardiac puncture and centrifuged, and the plasma was stored at –20°C. The experiments were approved by the University of Kuopio animal ethics committee.

Extraction of fecal samples was modified from a method developed in our laboratory (17). The samples were mixed with 2.5 volumes of 0.2 M sodium acetate in 10% NaCl, and aliquots (2 ml) were spiked with aflatoxin G₂ (AFG₂) (18.6 pmol/sample) as an internal standard and centrifuged (3,000 x g; 15 min; 4°C). The pellets were suspended in 4 ml 80% methanol (in 10% NaCl [vol/vol]) and homogenized thoroughly (MICCRA D-8; ART Labortechnik). Following a second centrifugation, the supernatant was reduced (under an N₂ stream at 50°C) and diluted with Milli-Q water, and aflatoxin residues were isolated using solid-phase extraction columns (Strata C18-E; 55 µm; Phenomenex). The eluates were reduced and diluted with Milli-Q water for immuno-affinity column (AflaTest; Vicam) cleanup. The cleaned-up samples were dried in vacuo, reconstituted in 500 µl methanol, and stored at –20°C for high-performance liquid chromatography (HPLC) analysis.

Urine samples were acidified with 0.1 N HCl to pH 5 and centrifuged (3,000 x g; 15 min; 4°C), and the supernatants were stored at –20°C. Aliquots (0.5 ml) were spiked with AFG₂ (9.3 pmol/sample), and the aflatoxin residues were isolated and concentrated as mentioned above. Reverse-phase HPLC conditions were used as described previously (17). AFB₁ and AFM₁ levels in urine and feces were corrected for intersample variation using AFG₂ as an internal standard and calculated from a standard curve in urine and feces as total daily excretion. Aflatoxin peak identities were confirmed by spiking the samples with the respective toxin standards and reanalysis by HPLC. The levels of AFB₁-albumin adduct were determined by albumin extraction, digestion, and enzyme-linked immunosorbent assay as previously described (2). Alanine transaminase (ALT) activity was measured in rat plasma using a commercial kit (Thermo Electron Corp., Mass.). The data were subjected to the Mann-Whitney U test using SPSS 11.5 for Windows.

In AFB₁-only treated rats, fecal excretion of both AFB₁ (15.6 ± 9.8 nmol/24 h feces) and AFM₁ (27.1 ± 22.5 nmol/24 h feces) were highly variable between animals but were positively associated (P < 0.004) within each animal. Their presence reflects unabsorbed aflatoxins, rather than systemic uptake and subsequent biliary excretion, since rat bile mainly contains conjugated rather than free aflatoxin metabolites (3, 13). The presence of AFM₁, a hydroxylated AFB₁ metabolite with considerable toxicity and carcinogenicity (14), in the feces suggests that this metabolite was formed by enterocytes, which are known to possess the necessary cytochrome P450 enzyme activity (4, 13), and diffused back into the intestinal lumen. Within 24 h following AFB₁ dosing, probiotic treatment significantly increased fecal excretion of AFB₁ by 122% (35.3 ± 21.3 nmol/24 h feces; P = 0.015) and of AFM₁ by 152% (68.1 ± 26.1 nmol/24 h feces; P = 0.001) (Fig. 1). These data suggest that GG was able to retain additional AFB₁ and AFM₁ inside the intestinal lumens of rats, most probably by binding both aflatoxins to the bacterial surface (6-12). Similar levels of fecal AFB₁ and AFM₁ in GG-treated and untreated animals were observed on the second and third days postdosing, suggesting that probiotic aflatoxin binding occurs immediately after administration of AFB₁ or formation of AFM₁.

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FIG. 1. Effects of Lactobacillus rhamnosus strain GG on daily fecal AFB1 and AFM1 excretion over 3 days following a single dose of AFB1 (1.5 mg or 4.8 µmol/kg). The values represent means ± standard deviations (n = 12 rats/group; *, P < 0.015).

Furthermore, we evaluated the urinary excretion of AFM₁ and plasma AFB₁-albumin adduct, both markers for reduced hepatic uptake of administered AFB₁. Urinary excretion of AFM₁ was highest during the 24 h following dosing and was comparable to values reported in the literature (13). However, urinary AFM₁ excretion was not significantly reduced by the presence of probiotic bacteria (74.5 ± 8.5 versus 79.5 ± 9.1 nmol/24 h urine without and with GG dosing, respectively).

AFB₁-albumin adducts were detected in the plasma of rats receiving AFB₁ and were highly variable. The mean levels of AFB₁-albumin adduct were on average lower in animals receiving AFB₁ plus GG than in those receiving only AFB₁ (13.8 ± 4.5 versus 19.5 ± 10.1 ng/mg albumin; P = 0.149; 29% reduction), though this was not statistically significant. These findings from urine and plasma samples may reflect a saturation of the hepatic metabolizing capacity within the dosing regime used.

To assess the hepatotoxic effect of AFB₁ dosing, the activity of ALT, an indictor of liver injury, was measured in rat plasma. The levels of ALT activity were comparable to the values reported in the literature for Wistar rats at a similar AFB₁ dose (15) and were significantly correlated with the AFB₁-albumin adduct levels (P = 0.004). ALT activity was increased by 149% in the group receiving AFB₁ alone (103.7 ± 84.9 U/liter; n = 9; P = 0.053) compared to the controls (41.6 ± 18.7 U/liter; n = 4). GG treatment reduced the AFB₁-induced increase in ALT activity (56.4 ± 34.2 U/liter; n = 9), though the difference was not statistically significant (P = 0.171). These findings indicate that the probiotic treatment increases aflatoxin retention within the gut and may consequently reduce the toxic effects of a high dose of AFB₁ in rats, though high individual variation in liver function and a small number of animals may have reduced the power of the study.

The body weights of animals at the beginning of our study were similar in all groups (156.0 ± 23.1, 155.0 ± 8.0, and 154.8 ± 7.9 g for untreated, AFB₁ only, and AFB₁ plus GG groups, respectively). Weight gains in untreated controls (8.2 ± 2.0 g/day) and animals in both treatment groups prior to AFB₁ dosing (7.0 ± 0.9 g/day, P = 0.211, and 7.2 ± 1.2 g/day, P = 0.332, for AFB₁ alone and AFB₁ plus GG, respectively) were not different (Fig. 2). Weight loss was observed in AFB₁-only-treated animals (–3.12 ± 2.94 g gain/day), whereas those also treated with GG showed an alleviation of this acute growth faltering (0.04 ± 2.19 g gain/day; P = 0.011) (Fig. 2), though they did not continue to grow at the rate observed prior to AFB₁ dosing. AFB₁-induced reductions in feed intake and body weight gain in rats in a dose-dependent manner were previously reported (16). Since probiotics alone had no effect on body weight gain, they may act by reducing the availability of free AFB₁ within the intestinal tract and thus reducing its toxicity.

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FIG. 2. Effects of Lactobacillus rhamnosus strain GG on daily body weight gain before and after a single dose of AFB1 (1.5 mg or 4.8 µmol/kg). The values represent means ± standard deviations (n = 4 rats/group for untreated controls and n = 12 rats/group for rats receiving AFB1 only or AFB1 and GG; *, the P value refers to the difference in body weight gain after AFB1 dosing between the AFB1-only and AFB1-plus-GG groups).

In conclusion, our data suggest that by increasing the excretion of orally dosed aflatoxin via the fecal route, probiotic treatment prevents weight loss and reduces the hepatotoxic effects caused by a high dose of AFB₁. However, further studies in which AFB₁ is administered repeatedly, mixed into feed and at naturally occurring levels, are needed before we fully understand the potential of probiotics to reduce absorption of AFB₁ for future use in a human AFB₁ exposure scenario.

 
* Corresponding author. Mailing address: Department of Clinical Nutrition, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland. Phone: 358-17 163615. Fax: 358-17 162792. E-mail: silvia.gratz{at}uku.fi.

Published ahead of print on 15 September 2006.

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Applied and Environmental Microbiology, November 2006, p. 7398-7400, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01348-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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