Lactobacillus rhamnosus Strain GG Modulates Intestinal Absorption, Fecal Excretion, and Toxicity of Aflatoxin B ₁ in Rats

Abstract

In this study, the modulation of aflatoxin B 1 (AFB 1 ) uptake in rats by administration of the probiotic Lactobacillus rhamnosus GG was demonstrated. Fecal AFB 1 excretion in GG-treated rats was increased via bacterial AFB 1 binding. Furthermore, AFB 1 -associated growth faltering and liver injury were alleviated with GG treatment.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 B 1 (AFB 1 ) and aflatoxin M 1 (AFM 1 ), 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 AFB 1 binding of this probiotic (6,7,11,12). GG was subsequently demonstrated to bind AFB 1 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 AFB 1 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 ϭ 12/group) received either vehicle (phosphate-buffered saline [PBS]) or probiotic suspension (5 ϫ 10 10 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 AFB 1 (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 beginning of the study (prior to GG treatment), on the day of AFB 1 dosing, and at the end of the study. Urine and fecal samples were collected daily, weighed, and stored at Ϫ20°C. At the end of the study, blood samples were taken by cardiac puncture and centrifuged, and the plasma was stored at Ϫ20°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 G 2 (AFG 2 ) (18.6 pmol/sample) as an internal standard and centrifuged (3,000 ϫ g; 15 min; 4°C). The pellets were suspended in 4 ml 80% methanol (in 10% NaCl [vol/vol]) and h...

Effect of oral supplementation of Lactobacillus reuteri in reduction of intestinal absorption of aflatoxin B1 in rats

“…According to this, the amount of adducts present in blood samples of rats treated only with AFB 1 represent the cumulative dose of aflatoxin intake over the experimental period, which indicates that the reduction of AFB 1 -Lys adduct observed in animals treated with aflatoxin plus bacteria was originated by the ability of L. reuteri to bind AFB 1 inside the intestinal lumen, thus avoiding its passage into the bloodstream. In a related work [13] no significant differences were found in the amounts of AFB 1 -Lys adduct present in animals receiving Lactobacillus rhamnosus GG daily for 3 d before and 3 d after a single oral dose of AFB 1 compared with those receiving only the mycotoxin. Other reports [11,30] suggested that probiotics are less capable of binding AFB 1 in the presence of mucus and are more susceptible to interfering factors in the intestinal tract, which may explain the behavior observed in the levels of adduct reported by Gratz et al [13].…”

“…In addition, ex vivo protocols [11] have also revealed that efficacy of AFs binding is variable depending on the genus and bacteria strain used [12]. Meanwhile, in vivo assays suggest that a mycotoxin single dose may not represent the complete picture of potential of probiotics to reduce absorption of AFB 1 during a chronic exposure to aflatoxins [13]. A previous work using L. reuteri NRRL14171 [14] proved that this strain not only binds AFB 1 efficiently in vitro, but also possesses comparable binding ability with other strains previously reported [8,11].…”

Aflatoxicosis: Lessons from Toxicity and Responses to Aflatoxin B1 in Poultry

“…However, most research has focused on probiotic strains of Lactobacillus, Bifidobacterium, and Propionibacterium [42,266,267,[270][271][272][273][274][275][276][277][278][279][280][281][282][283][284][285][286][287][288]. Interactions between AFB1 and Lactobacillus rhamnosus GG (LGG), L. rhamnosus LC-705 (LC-705), Propionibacterium freudenrieichii strain shermanii JS (PJS) or mixtures of these strains have been shown to be especially effective [42,[270][271][272][273][274][276][277][278][279][280][284][285][286]288]. A mixture of these strains was also utilized in our transcriptomic analyzes [238,240].…”

“…Reduced AFB1 toxicity has been demonstrated in both mice [270] and in rats [277] after addition of dietary LGG. In humans, probiotics enhanced excretion of AFB1 [289,290].…”

Protective Effects of Lactobacillus rhamnosus GG on Aflatoxins-Induced Toxicities in Male Albino Mice

“…Previous work showed that LGG was the most efficient strain in binding a range of mycotoxins, including aflatoxins [27]. Furthermore, Gratz et al [90] suggested that LGG treatment reduced the hepatotoxic effects caused by a high dose of AFB 1 , by increasing the excretion of orally dosed aflatoxin via the fecal route and suggested that LGG was able to retain additional AFB 1 and AFM 1 inside the intestinal lumens of rats. Moreover, LAB found to cause reduction of the formation of secondary bile acids [91] and enhancement of the immune system [92][93][94].…”