Probiotic Pediococcus pentosaceus strain GS4 alleviates azoxymethane-induced toxicity in mice

Abstract

Probiotic treatment has been gaining attention due to its remarkable effects in alleviating toxicity and carcinogenesis. The novel strain Pediococcus pentosaceus GS4 has been reported for probiotic, survivability in simulated gastrointestinal fluid, and antioxidative and biohydrogenation properties. Therefore, we hypothesize that this specific strain might be able to assuage the effect of azoxymethane (AOM)-induced toxicity in mice. Twenty-eight Swiss albino mice were divided into 4 groups and were studied for 32 weeks. Azoxymethane (10 mg/kg body weight) was administered intraperitoneally twice (0^th and 14^th days), and probiotic GS4 (1.1 × 10⁹ colony-forming unit/mL) was given orally for the respective groups. Mice who served as the normal control received only normal saline. GS4-intervened AOM-induced mice showed marked improvement at the histopathologic level, in the liver and kidney. Moreover, probiotic GS4 intervention in AOM-induced mice exhibited a significant reduction in the liver function biomarker when compared with the AOM-induced mice. Probiotic GS4 intervention reduced the intestinal structural deformities as evident from the elevated brush border membrane–associated disaccharidases (sucrase, lactase) and intestinal alkaline phosphatase activities, which were found disrupted by AOM intoxication. Fecal bacterial load was found to be reduced in AOM-induced mice which were subsequently replenished by the probiotic GS4 intervention as apparent from the enhanced fecal bacterial load. There were no adverse effects observed in the probiotic control group. Conclusively, novel probiotic strain GS4 exhibited safe and beneficial effects against the toxicity threats posed by AOM. Thus, GS4 could be considered as a potential food supplement/additive for therapeutic purposes in gastrointestinal disorders related to inflammation and cancer.

Introduction

Colorectal cancer is the third most common form of cancer, making up about 10% of all cases [1], and is known to be triggered by adenomatous polyposis generated in the colon due to distinct factors, including diet and chemical intoxication [2], [3], [4]. Different inflammation-related diseases like inflammatory bowel disease, Crohn disease, and ulcerative colitis are considered high-risk factors for the development of colon cancer, which may subsequently metastasize into the liver and kidney [5], [6]. Although different targeted therapies including chemotherapies are available for colon cancer and inflammatory diseases [7], their uses get relatively restricted because they are generally associated with gastrointestinal adverse effects [8]. Moreover, antibiotics that are used to overcome the gastrointestinal adverse effects associated with chemotherapy also have adverse effects on gut microbiota [9], so there was a necessity to develop a novel therapeutic approach that can avoid these adverse effects on the gut microbiota, combat cancer, and also ameliorate the probability of prognosis in the secondary target organs. In recent times, the probiotic therapy has been gaining a lot of attention as an active research front for the mitigation of toxicity generated by the carcinogenic azoxymethane (AOM).

Azoxymethane intoxication generally occurs in the liver, kidney, and intestine as secondary targeted organs [10] and is widely used to generate multiple-organ toxic models in rodents [11]. Azoxymethane is metabolically activated with hydroxylation of a methyl group by cytochrome P450 2EI to produce methyl azoxymethanol which is further transformed to a highly reactive alkylating species methyl diazonium, which causes inflammation [12]. Many studies have shown that the AOM model could also be used as a hepatotoxic model, where acute liver failure occurs. This was examined by the elevation of hepatic enzymes such as alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) [13]. Besides liver failure, inflammation caused by AOM intoxication can lead to structural deformities and impaired brush border membrane (BBM) in the intestine; this can often result in insufficient production of intestinal disaccharidases (sucrase, maltase, and lactase) and intestinal ALP (i-ALP) [14], [15]. Furthermore, the deregulation of disaccharide metabolism in the intestine leads to a condition called malabsorption [15]. There is enough scientific evidence to support the fact that AOM toxicity reduces the microbial load in the gut and thus leads to dysbiosis [16], [17]. Sustained dysbiosis leads to many systemic diseases and disorders, including diabetes, obesity, irritable bowel syndrome, and colon cancer [17]. Thus, if the gut microbial load could be elevated in these conditions by probiotic intervention, then there could certainly be a mitigation of these inflammatory processes.

Probiotic is an important component of functional food which offers several benefits to the host either by reducing the impact of carcinogens or through xenobiotic metabolism or by producing health-promoting metabolites. Probiotic cures different types of liver diseases without causing adverse effects [18]. Studies have shown probiotics (eg, Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii subsp Shermanii) in the hepatoprotective role which can symbiotically reduce the toxicity generated by aflatoxin in the liver [19]. Similarly, Enterococcus lactis IITRHR1 has demonstrated its potential in lowering the level of different serum biomarker enzymes in acetaminophen-induced hepatotoxicity [20]. The probiotic L rhamnosus GG showed its potential in modulating BBM-associated disaccharidases and i-ALP in the inflamed intestine caused by Giardia intestinalis [21]. In addition, Ochratoxin A–mediated toxicity could also be ameliorated by the intervention of Lactobacillus reuteri CRL 1098 and Lactobacillus acidophilus CRL [22]. Moreover, it was found that probiotics can also replenish the altered microbial load and contribute to maintenance of structural integrity and hence can be used for the establishment of the intestinal barrier [23]. Previous studies demonstrated that probiotics can efficiently protect the intestine from chemical intoxication by reducing the oxidative stress and also by secreting different antioxidants. In our earlier study with Pediococcus pentosaceus strain GS4, it was observed that GS4 efficiently produced antioxidants and conjugated linoleic acid; a health beneficial metabolites by biohydrogenation and also able to survive in simulated gastrointestinal fluids [24], [25], [26], [27]. Therefore, we were interested to test the hypothesis of whether this probiotic strain GS4 might also have the ability to mitigate the toxic effects of a chemical carcinogen with the modulation of the gut microbiota. To test our hypothesis, we first evaluated the protective efficacy of probiotic GS4 at the structural level through histologic analysis of the liver and kidney, whereas liver functionality was determined biochemically. In order to demonstrate the shielding effect of the probiotic on the intestinal structural integrity, the biomarkers related to intestinal structure and function were also studied. This study also aimed to ascertain the precise role of probiotic GS4 in the alleviation of induced toxicity through replenishing the microbial load in the gastrointestinal tract disrupted by chemical carcinogens.