Lactobacillus rhamnosus Strain GG Modulates Intestinal Absorption ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2006, p. 7398–7400 0099-2240/06/$08.00⫹0 doi:10.1128/AEM.01348-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 72, No. 11

Lactobacillus rhamnosus Strain GG Modulates Intestinal Absorption, Fecal Excretion, and Toxicity of Aflatoxin B1 in Rats䌤 S. Gratz,1,2* M. Ta¨ubel,2 R. O. Juvonen,3 M. Viluksela,4 P. C. Turner,5 H. Mykka¨nen,1 and H. El-Nezami2 Department of Clinical Nutrition, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland1; Food and Health Research Centre, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland2; Department of Pharmacology and Toxicology, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland3; National Public Health Institute, Laboratory of Toxicology, P.O. Box 95, 70701 Kuopio, Finland4; and 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

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.

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 ⫻ 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 ␮m; 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 ␮l methanol, and stored at ⫺20°C for highperformance liquid chromatography (HPLC) analysis. Urine samples were acidified with 0.1 N HCl to pH 5 and centrifuged (3,000 ⫻ 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 enzymelinked immunosorbent assay as previously described (2). Alanine transaminase (ALT) activity was measured in rat plasma

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 ⫻ 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 ␮mol/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

* 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 @uku.fi. 䌤 Published ahead of print on 15 September 2006. 7398

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PROBIOTICS MODULATE AFLATOXIN ABSORPTION AND TOXICITY

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 ␮mol/kg). The values represent means ⫾ standard deviations (n ⫽ 12 rats/group; *, P ⬍ 0.015).

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. 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

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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 ␮mol/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 AFB1only and AFB1-plus-GG groups).

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-onlytreated 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. 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

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