Gut microbiota limits heavy metals burden caused by chronic oral exposure

Highlights

• We model chronic ingestion of environmental lead and cadmium in axenic mice.

• We addressed the role of the microbiota in heavy-metal dissemination in organs.

• We delineate the direct impact of the non-absorbed heavy metals on gut homeostasis.

• We measure transport- and oxidative-gene expression in intestine.

• It enlightens risk assessment of heavy metals in intestinal disease's susceptibility.

Abstract

Environmental exposure to pollutants such as heavy metal(s) is responsible for various altered physiological functions which are detrimental for health. The gut microbiota is critical for intestinal homeostasis but its role on xenobiotic handling is not fully understood, especially when continuous sub-chronic exposure is addressed. We first confirmed the essential role of the intestinal microbiome to limit heavy metal body burden by using germ-free mice following 6-weeks oral exposure. Significant increases of cadmium and lead absorption and dissemination in blood and target organs were measured in germ-free mice when compared with conventional specific pathogen free (SPF) mice. Besides the “barrier” function of the luminal microbiota, this may involve specific host-genes such as metallothioneins, which are differentially expressed in the gastrointestinal tract of each group of mice. Considering genes relevant for divalent metal transporters and oxidative pathways, significant differences in basal gene expression were measured between control and germ-free mice. Moreover, the magnitude of induction of these genes upon stimulation by heavy metals varied greatly depending on the dose and type of metal as well as the microbial status of the animal. Collectively, these data illustrate the complex host-microbes interplay occurring with environmental pollutants inside the gut.

Introduction

Inorganic cadmium (Cd) and lead (Pb) ions are the most representative toxic non-essential elements which can contaminate food, water or air. In industrial areas, occupationally exposed workers as well as environmentally exposed populations can experience moderate to severe health perturbations due to the chronic ingestion of heavy metals. It has been shown that a significant fraction of inhaled Cd (60%) ends up in the gastrointestinal tract, as a result of mucocilliary clearance and subsequent ingestion (Satarug et al., 2003). Following oral entry of Cd and Pb, the body burden of these metals has been clearly linked to various sorts of diseases based on various mechanisms, including those involved in oxidative stress and extended toxicity such as genotoxicity and carcinogenesis.

The impact of gut ecology on the absorption, distribution, metabolism and excretion of xenobiotics has received little attention (Nicholson et al., 2005). However, the gut microbiota is likely an important mediator of the bioavailability and toxicity of environmental pollutants including heavy metals. On the one hand, the microbiota by itself may interact with metals inside the gut, either by active uptake or by passive ad- or absorbtion (Morozzi et al., 1986, Halttunen et al., 2008). On the other hand, intestinal barrier integrity, as the first line to control the entry of ingested toxic metals, also depends on microbial-host interactions that involve epithelial junctions and physical impediments of the mucous layer. Finally, the gut microbiota and its metabolites will also impact on environmental parameters such as pH, oxidative balance, detoxification enzymes, and xenobiotic-metabolizing and transporting host proteins (Claus et al., 2011), all of which may highly influence the bioavailability of chemicals in the gut lumen. The microbial status furthermore affects hepatic and renal metabolism.

While Louis Pasteur considered that germ-free life was impossible (Pasteur, 1885), today we know that germ-free mice are available and can be grown as far as proper technical requirements associated with rearing are achieved; the concept of the gut microbiota as a “forgotten organ”, essential for gut homeostasis, is also well accepted. Therefore, axenic mice, so called germ-free mice provide useful tools to study the interplay between the host and the gut inhabitants (Smith et al., 2007, Yi and Li, 2012). Germ-free animals are, however, physiologically different from conventional specific pathogen free (SPF) individuals and thus may show differences in the in vivo handling of xenobiotics (Ilett et al., 1990).

Recently, we reported on the dissemination to the intestine and to primary organs of continuous ingestion of low- and moderate environmentally relevant concentrations of Cd and Pb in mice (Breton et al., 2013). The aim of the current study was to address the role of the gut microbiota in the bioaccumulation and retention of Cd and Pb in primary organs following oral exposure. To examine these events, we used germ-free and SPF mice subjected to a chronic ingestion of various environmentally relevant doses of Cd and Pb. We also determined changes in gene expression of several intestinal markers, addressing aspects of transport- and oxidative stress functionality of the proximal (duodenum) and distal (colon) mucosa.