The unrecognized occupational relevance of the interaction between engineered nanomaterials and the gastro-intestinal tract: a consensus paper from a multidisciplinary working group
Pietroiusti et al. Particle and Fibre Toxicology
The unrecognized occupational relevance of the interaction between engineered nanomaterials and the gastro-intestinal tract: a consensus paper from a multidisciplinary working group
Antonio Pietroiusti 0 1
Enrico Bergamaschi 3
Marcello Campagna 2
Luisa Campagnolo 1
Giuseppe De Palma 7
Sergio Iavicoli 6
Veruscka Leso 5
Andrea Magrini 1
Michele Miragoli 4
Paola Pedata 8
Leonardo Palombi 1
Ivo Iavicoli 0 5
0 Equal contributors
1 Department of Biomedicine and Prevention, University of Rome Tor Vergata , Via Montpellier 1, 00133 Rome , Italy
2 Department of Medical Sciences and Public Health, University of Cagliari , Cagliari , Italy
3 Department of Sciences and Public Health and Pediatrics, University of Turin , Turin , Italy
4 Department of Medicine and Surgery, University of Parma , Parma , Italy
5 Department of Public Health, University of Naples Federico II , Naples , Italy
6 Department of Occupational and Environmental Medicine, Epidemiology and Hygiene, Italian Workers' Compensation Authority (INAIL) , Rome , Italy
7 Department of Medical and Surgical Specialties, Radiological Sciences, and Public Health, Section of Public Health and Human Sciences, University of Brescia , Brescia , Italy
8 Department of Experimental Medicine- Section of Hygiene, Occupational Medicine and Forensic Medicine, University of Campania Luigi Vanvitelli , Naples , Italy
Background: There is a fundamental gap of knowledge on the health effects caused by the interaction of engineered nanomaterials (ENM) with the gastro-intestinal tract (GIT). This is partly due to the incomplete knowledge of the complex physical and chemical transformations that ENM undergo in the GIT, and partly to the widespread belief that GIT health effects of ENM are much less relevant than pulmonary effects. However, recent experimental findings, considering the role of new players in gut physiology (e.g. the microbiota), shed light on several outcomes of the interaction ENM/GIT. Along with this new information, there is growing direct and indirect evidence that not only ingested ENM, but also inhaled ENM may impact on the GIT. This fact, which may have relevant implications in occupational setting, has never been taken into consideration. This review paper summarizes the opinions and findings of a multidisciplinary team of experts, focusing on two main aspects of the issue: 1) ENM interactions within the GIT and their possible consequences, and 2) relevance of gastro-intestinal effects of inhaled ENMs. Under point 1, we analyzed how luminal gut-constituents, including mucus, may influence the adherence of ENM to cell surfaces in a size-dependent manner, and how intestinal permeability may be affected by different physico-chemical characteristics of ENM. Cytotoxic, oxidative, genotoxic and inflammatory effects on different GIT cells, as well as effects on microbiota, are also discussed. Concerning point 2, recent studies highlight the relevance of gastro-intestinal handling of inhaled ENM, showing significant excretion with feces of inhaled ENM and supporting the hypothesis that GIT should be considered an important target of extrapulmonary effects of inhaled ENM. Conclusions: In spite of recent insights on the relevance of the GIT as a target for toxic effects of nanoparticles, there is still a major gap in knowledge regarding the impact of the direct versus indirect oral exposure. This fact probably applies also to larger particles and dictates careful consideration in workers, who carry the highest risk of exposure to particulate matter.
Ingested nanoparticles; Inhaled nanoparticles; Direct toxicity; Indirect toxicity; Workers' exposure; Gastrointestinal tract; Microbiota
Despite the large and growing number of ENM used in
agri-food products [
], oral ingestion has received
significantly less attention than the pulmonary route and
therefore there is relatively lower information on the
possible toxic effects of ENM on the gastro-intestinal
tract (GIT). This may be due to the fact that the study
of the impact of ENM on the GIT (and vice versa) is a
rather complicated issue: both food and the processes
that break down and transform food ingredients (e.g.,
physical forces, osmotic concentration and pH gradients,
digestive enzyme, redox conditions and salinity levels)
may in fact transform, aggregate and dissolve ENMs in
ways that alter their naive and inherent properties,
therefore potentially affecting their biological reactivity
as well as their toxicological profiles.
This picture is however changing: It is becoming clear
that the gut micro-organisms (the microbiota) play a
pivotal role in maintaining both local (intestinal) and
systemic homeostasis and that they may influence ENM
and be influenced by them [
]. Very recent in vitro and
in vivo data, discussed in the first section of the present
review, have shown that ingested ENM may induce
substantial adverse effects unrecognized in past studies; last
but not least, there is indirect growing evidence that
inhaled ENM, representing the most common pathway of
exposure in workers, may have a substantial impact on
the GIT, as shown in the second section of the review.
In September 2016, the Italian Society of
Occupational Medicine and Industrial Hygiene (SIMLII) hosted
a research workshop in order to exchange and merge
knowledge and expert point of view on the
abovementioned topics. In the following sections, we outline
how these topics have been developed and summarize
the state of the evidence about their possible impact on
future research in the field of nanotoxicology.
Interaction of ingested ENM with the GIT
Aggregation, agglomeration and dissolution
The fate and bioavailability of ENM in the
gastrointestinal system may be affected, at least partly, by their
primary characteristics, such as size, surface chemistry and
charge, or, in turn, by properties acquired through the
transit via the GIT. Several factors, such as pH gradients,
gastrointestinal transit time, nutritional status, meal
quality, level of mucosal and enzymatic secretions, as
well as the intestinal microflora, may all influence ENM
physical and chemical reactivity [
]. There is limited
information on the physical changes of some metallic
ENM (Ag, TiO2, SiO2 and ZnO) once in contact with
the gastro-intestinal fluids. It seems that ion release may
occur in the gastric environment, along with
sizedependent aggregation and agglomeration. For example,
it has been shown that in gastric juice ZnO and Ag
undergo dissolution [
]. Agglomeration has been shown
for TiO2 and also for Ag ENM, dependently from size
9–13, 15, 16
]. Conflicting data have been obtained as
far as aggregation/agglomeration in the intestinal
environment is concerned: agglomeration has been reported
for SiO2  and de-agglomeration for Ag [
the chemical composition of ENM, their surface charge
and the fasting/fed state may be important components
of the final outcome. Clearly, more data are needed in
order to understand how different variables such as
previous transit in different environments, fasting and fed state
may each contribute to the final physical status of ENM
travelling along the GIT. In addition, a higher number of
ENM and of gastro-intestinal physiological states should be
ENM uptake and absorption
Although limited information is available on the
toxicokinetics of orally administered ENM [
], available data
suggest that uptake and absorption of ENM in the GI
tract may have relevant implications for their local and
systemic effects [
A detailed description of the GIT cellular and
extracellular structures involved in the uptake and absorption of
ENMs, and of the mechanisms of uptake are beyond the
scope of this review, however a brief presentation of the
main players is needed in order to understand the fate of
ENM in the intestine.
In this regard, the key cell types are a) the enterocytes,
which are by far the most represented cell type along the
intestine and are connected each other by tight junctions,
which prevent the unselected intercellular access to the
luminal content; b) the antigen sampling M cells, overlying
organized lymphoid structures such as the Peyer’ s patches
and other gut-associated lymphoid tissue (GALT).
Although representing only 1% of the intestinal cells, M cell
are covered by a much thinner mucus layer than
enterocytes, and are very relevant for the uptake of foreign
substances, which are subsequently delivered to the underlying
lymphoid cells; c) the mucus producing goblet cells (about
10% of the total intestinal cells), secreting the mucus lining
the whole surface of the small and large intestine.
The first barrier encountered by ingested ENM is
indeed represented by mucus, which has been reported to
be efficient in trapping larger ENM [
], this factor
being a possible explanation for the less pronounced toxic
effects of 200 nm Ag ENM in comparison to 20 nm Ag
ENM observed in in vitro experiments on a co-culture
of CaCo 2 cells and mucus producing cells [
Ingested ENM may on the other hand influence mucus
secretion, in both quantitative and qualitative terms.
For example, sub-chronic (28-days) oral exposure to
60 nm Ag ENM in rats [
] promoted the secretion of
mucus in the ileum and rectum, and changes in mucin
composition (amounts of neutral and acidic mucins
and proportions of sulfated and sialylated mucins). This
may be interpreted as a non-specific inflammatory
Once crossed the mucus barrier, ENM come in
contact with the intestinal cells: the main mechanism
through which they may cross the intestinal barrier is
represented by transcellular transport. Available in vitro
studies suggest that smaller particles may traverse
enterocyte cell membranes, mediating changes in
membrane fluidity, resulting in altered signaling or increased
permeability and cytotoxicity; conversely, as particle
size increases or as agglomeration occurs, uptake is
predominantly performed by M cells, which are already
specialized for this function [
]. Of note, the
immunologic responses by lymphoid tissue beneath M cells is
typically oriented to hypo-responsiveness (oral
tolerance). It is not known, however, whether environmental
ENM can have similar mucosal immunologic effects.
Evidence for this possibility arises from the observation
that agglomerates of endogenous calcium-phosphate
nanoparticles (of similar size to ENM in biological
media) and dietary TiO2 can bind gut microbial-derived
molecules (e.g. peptidoglycan, lipopolysaccharide) and
traffic these to GALT, with influence on tolerance or
In vivo studies in rodents suggest that a low percentage
of ENM present in the gut lumen is actually absorbed. For
example, in a long term study (24 or 84 days) of orally
administered amorphous silica (7 nm or 10–25 nm),
absorption was 0.25% [
]. A higher uptake was reported in
another 10 day administration study, in which 500 nm
TiO2 particles, given by gavage, were taken up in
percentages ranging from 0.11%, in the stomach, to 4% in the
large intestine, and the vast majority of the ENM
accumulated in the Peyer’s patches [
]. Although the size of
TiO2 particles in the above-mentioned study was beyond
the conventional size limit of ENM (100 nm), the findings
of another report, showing the presence of 12 nm TiO2
particles in Peyer’s patches soon after a single
administration by gavage, suggest that early absorption of ENM
]. Of relevance, available information from in vitro
experiments suggests that uptake of ENM may be
decreased by food components, as shown for silica and
]. Some data regarding TiO2 upake and
absorption, after a single administration, are available also
for humans, and they range from no evidence of
absorption (TiO2 size: 10–1800 nm; dose range 315–620 mg)
] to detectable elemental Ti in blood after the
administration of 100 mg of 260 nm particles [
Data regarding chronic low dose exposure are of
course needed in order to clarify the presence and extent
of intestinal absorption in humans under real life
In the following paragraphs the most relevant available
in vitro and in vivo studies on ENM toxicity on the GIT
In vitro studies
Cell damage In Caco-2 cells, TiO2 ENM exposure
caused loss and morphological changes in microvilli and
disorganization of the brush border [
rutilecored aluminum hydroxide and
polydimethylsiloxanesurface treated TiO2 ENM did not cause any damage
]. Epithelial alterations, consisting of plasma
membrane disruption and tight junction loosening, have been
demonstrated also by Mahler et al. [
] in a tri-culture
gut model including enterocytes, goblet cells and M
cells, treated with 50 and 200 nm polystyrene beads.
Changes in permeability Changes in permeability of the
epithelial barrier may be interpreted as the result of
functional damage to the integrity of the intestinal barrier,
sometimes preceding the development of evident cellular
damage. Ag ENM treatment of T84 human colonic
epithelial cells, characterized by polarized monolayers
naturally producing mucus, induces size- and dose-dependent
changes in the expression of genes involved in anchoring
tight junctions, which results in increased intestinal
]. A significant increase in epithelial
permeability of Caco-2 tight monolayers was reported also for
TiO2 ENM [
] and a reversible effect was also observed
for differently functionalized fullerenes and single walled
carbon nanotubes (SWCNTs) [
However, other studies performed on Caco-2 monolayers
as well as on Caco-2/HT29-MTX co-culture models failed
to detect such alterations both for TiO2 [
], SiO2 [
Ag ENM [
These conflicting findings may be explained by the
different doses, in vitro models, methods of detection and
physico-chemical characteristics of the tested ENM. As in
other experimental settings regarding the study of toxicity
of ENM, grouping of ENM and standardized experimental
conditions may help to clarify the role of different ENM in
inducing alterations of intestinal permeability.
Cell viability and proliferation Different culture
methods have been used in order to study the effects of
ENM on viability of cells of the GIT. Since different models
may show different sensitivity, we are presenting separately
data regarding undifferentiated and differentiated
monocultures and those regarding co-culture models.
Studies in undifferentiated mono-cultures These
studies generally show alterations in cell viability induced by
metal based ENM, with high cytotoxicity induced by
], SiO2 [
17, 44, 45
], and Ag ENM [
Milder cytotoxic effects have been reported for Au
], TiO2 ENM [
15, 32, 33, 40, 48, 50–56
carbon nanotubes [
37, 51, 57, 58
] in short-term studies.
Studies in fully differentiated Caco-2 cells cultures
Relatively few studies are available in fully differentiated
Caco-2 cells, which, however, better reflect the native
GIT and are generally less sensitive to cytotoxic injuries
]. Nevertheless, some ENM such as Ag and ZnO
ENM  are equally toxic to both undifferentiated and
differentiated cultures. It remains to be defined whether
the effect is attributable to ENM themselves, or to ion
release or to both [
Studies in co-culture models Toxicity of nanomaterials
(TiO2 NM101, Ag NM300, Au) has been evaluated in
non-inflamed and inflamed co-cultures, and also
compared to non-inflamed Caco-2 monocultures. The
inflamed co-cultures released higher amounts of IL-8
compared to Caco-2 monocultures, but the cytotoxicity
of Ag NP was higher in Caco-2 monocultures than in
3D co-cultures [
]. However, other investigations
failed to detect such differential vulnerability of Caco-2
monocultures to Ag ENM [
22, 39, 61–64
Ag ENM were found to be more toxic than TiO2 or Au
ENM , while negligible toxic effects have been
reported for Carbon nanotubes [
]. More complex
in vitro intestinal models have been proposed, such as
organoid cultures; these seem very promising for
studies on diseased gut, however such models are not
completely characterized yet .
When investigating in vitro the potential toxicity of
ENM on the GIT, several in vivo occurring phenomena
should be considered and reproduced to more faithfully
mimic the in vivo conditions. As already discussed, there
is an ongoing debate on the contribution of the
timedependent dissolution and ion release from metal based
ENM to cytotoxic effects [
Moreover, the modifications induced to ENM after
the interactions with different GIT compartments
should be carefully addressed. For instance, acid
treatment simulating quantum dots (QD) exposure to
gastric juice increased the toxicity of PEG-coated QDs on
Caco-2 cells, as a consequence of coating removal,
which enabled dissolution into Cd2+ ions [
Conversely, simulation of Ag ENM digestion, with or
without organic and food components, did not significantly
affect cytotoxicity and only caused minor
agglomeration of particles [
]. Therefore, considering that
pH in vivo varies across different gut compartments
and with composition of ingesta, future research should
aim to clarify whether and to what extent these
conditions may affect ENM toxicity. The effect of food
should also be considered. A paradigmatic example is
that of micronutrients; whereas phenolic compounds
(namely, quercetin and kaempferol), present in fruits
and vegetables, can protect Caco-2 cells from Ag ENM
induced toxicity and thus maintained the integrity of
the epithelial barrier, resveratrol do not exert such
]. This protective action may be attributed
to the potent anti-oxidant properties of flavonoids.
Another study failed to detect differences in cytotoxicity
between digested or undigested Ag ENM on Caco-2
cells when the digestion process was implemented with
the presence of the main food components, i.e.
carbohydrates, proteins and fatty acids [
]. Native TiO2
ENM and TiO2 ENM pretreated with digestion
simulation fluid or bovine serum albumin did not show
significant different toxicity in Caco-2 cells . The
administration of ZnO NPs in combination with fatty
acids, on the other hand, increased their cytotoxic
]. Overall, these data strongly support the
relevance, when investigating the potential toxicity of orally
ingested ENM, of developing in vitro models which
take into account the possible ENM transformation
after contact with food or food components, with acidic
pH, and GIT constituents in order to mimic in vivo
Studies regarding effects of ENM on cell viability are
detailed in Table 1, where doses, and physico-chemical
characteristics of tested ENM are also reported. For a
more complete overview of the available literature, the
table includes also studies that have not been discussed
in the text.
Overall, in vitro data are seemingly discordant, however,
as discussed above, different experimental conditions
(doses, exposure duration, cell types, functionalization)
may at least in part explain the different results. An
important potential causal factor is represented by the
effective dose cells are challenged with, which may be quite
different in experiments using the same nominal dose:
ENM administration under static conditions to cells
cultured at the bottom of a culture plate may lead to different
interaction rate of the materials with the medium and
therefore with cells in different experiments, leading to
different cellular concentrations. For example, a fraction
of ENM may aggregate in liquid suspension and come in
contact with cells at relatively fast rate, whereas those
suspended may remain in suspension for the whole duration
of the experiment and never get in contact with the
cellular surface. Even small changes in the proportion between
aggregated and suspended ENM may lead to quite
different dose-response curves. The ultimate fate of ENM in a
fluid is then dictated by its mass density, i.e. nanomaterials
will settle if their mass density is greater than that of the
]. Suggestions to overcome these limitations have
recently been discussed [
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In vivo studies
Only a few studies have investigated toxicity of ENM in
vivo. Studies focusing on Ag ENM provided evidence for
liver inflammatory infiltration after acute and chronic
], although studies demonstrating no
toxic effects have also been reported [
the difference is mainly related to the dose used. Indeed,
in one of the studies demonstrating ENM adverse effects
on the GIT , a NOAEL (no observable adverse effect
level) of 30 mg/kg and LOAEL (lowest observable adverse
effect level) of 125 mg/kg were calculated. In studies
showing no toxicity, doses lower than the calculated
LOAEL were used.
Interestingly, in positive studies, the liver damage
was elicited at comparatively lower doses in mice
than in rats.
TiO2 ENM were found to induce inflammatory
changes in the small bowel [
] and also to enter the
systemic circulation to accumulate and cause
inflammation and oxidative damage in the liver, kidney and spleen
]. However, other studies did not detect any
adverse effect after oral administration of titanium dioxide,
even at very high doses [
In order to reconcile the contradictory data regarding
TiO2 ENM, Warheit and Donner [
] noted that
negative studies had been performed according to OECD
test guidelines, whereas those showing adverse effects
were “experimental-type” studies, and highlighted the
predominant use of mice in studies indicating adverse
effects and of rats in those showing no effects,
suggesting that differences in susceptibility of exposed animals
may contribute to the final result. In addition,
commercial test materials were used in studies showing no
effects, whereas “home-made” particles were more often
used in studies in which adverse effects were observed.
However, the presence of substantial adverse effects at
doses as low as 1 mg/kg/bw reported by Tassinari et al.
], and their absence at doses three order of
magnitude higher reported by Warheit et al. [
hard to be explained.
Local intestinal damage was reported after oral
ingestion of Carbon nanotubes (CNTs). Indeed, multiple
necrotic foci in the small intestine were observed after
a 30-days treatment with multi-walled carbon
nanotubes (MWCNTs) in mice, maybe related to the direct
CNT-mediated mechanical damage to the enterocytes
]; whereas a 6 month chronic exposure to MWCNTs
in rats induced a dose-dependent decrease in the
number of villi in the small intestine characterized by apical
]. Ingested ZnO ENM were reported to
undergo size-dependent intestinal absorption with
accumulation in multiple organs and damage to liver and
]. Finally, ingested SiO2 ENM caused
low-level hepatotoxicity in rats following a 10-week
exposure . As highlighted above, no observed
adverse effect levels (NOAEL), which might be
extrapolated to exposed workers, were calculated for some of
the studies following OECD guidelines [
73, 87–89, 92
for silver ENM a NOAEL of 30 mg/kg per day was
], whereas for ZnO the calculated
NOAEL was 268 mg/kg. NOAEL ranging from
1000 mg/kg to 24,000 mg/kg have been proposed for
titanium dioxide [
Mechanisms of toxicity
Mechanisms of ENM induced toxicity have been recently
] and will not be reported in detail here.
We will, however, discuss two developing new fields
represented by the interaction of ENM with the gut microbiota
and by the contribution that the “omics” technique may
give to detect effects which are not observed by using
traditional approaches. In the second section of this
review, we will also discuss the possible different toxicity
mechanisms occurring after direct ingestion of ENM or
indirect ingestion, following ENM inhalation.
Effects on intestinal microbiota
Most of the functions of the gastrointestinal tract are
facilitated, influenced or modulated by the vast resident
collection of microbes, known collectively as the
intestinal microbiota [
]. The intestinal microbiome has
been a major topic of research in the fields of
microbiology and medicine [
46, 99, 100
] and only recently it has
been considered in the context of potential toxicological
effects of ingested metals, including their nanoforms [
]. Given that a disruption of the normal intestinal
microbiota, also known as dysbiosis, has been linked to
severe medical conditions like colitis, inflammatory
bowel disease, diabetes and metabolic syndrome,
determining whether ENM have an impact on commensal
gut microbiota is an essential step in evaluating their
overall safety .
Few data are available from human. For instance, Das
et al. [
] found that the human microbiota (evaluated
in stool samples) could be significantly impacted in
metabolic activity, as demonstrated by the reduced total
gas produced by the stool microbial ecosystem as well as
in phylogenetic assemblages, since the anaerobe, Gram
negative abundance was significantly reduced by a
subacute 48 h exposure to 25–200 μg/ml Ag ENM.
Studies in rodents evaluated the effects of ingested Ag
ENM on the gut microbiota, although with non-univocal
]. Williams et al.  reported a
significant decrease in colony-forming units of indigenous ileal
microbial populations of rats sub-chronically gavaged
with 10–110 nm PVP-coated Ag ENM at doses of 9, 18
and 36 mg/kg bw/day for 13 weeks. The most
pronounced effects on cultivable bacteria were observed at
lower doses and with smaller diameter particles.
Importantly, when real-time PCR was utilized to amplify DNA
extracts, i.e. 16 s universal bacterial gene, to measure the
relative expression of bacteria, no significant differences
could be detected in any of the treatment groups. This
may be due to the fact that 16 s–based real-time PCR
technique, although proposed as the most suitable
method for the quantification of specific microbial
communities compared to the traditional culture strategy or
the next generation sequencing, is not able to distinguish
live bacteria from uncultivable dead or non-proliferating
microbes. Therefore, caution should be paid in the
interpretation of such kind of data. They also compared the
ratio of Bacteroidetes to Firmicutes, the two major phyla
of the intestinal microbiome, showing that 110 nm Ag
ENM at the highest dose induced a significant increased
ratio due to a decrease in Firmicutes. However, no clear
description was available concerning the physiologic
effect, either detrimental or beneficial, of these alterations
Another in vivo study [
] performed in mice,
showed that Ag ENM could affect the gut microbiota at
doses relevant for human dietary exposure (0.046–
4.6 mg/kg). In fact, a 28 day oral exposure to Ag ENM
mixed in food increased the ratio between Firmicutes
and Bacteroidetes phyla inducing a dose-dependent
decrease in Bacteroides and an increase in Firmicutes as
assessed by the next generation sequencing technique
]. The trend in Firmicutes alterations reported in
this study [
] was different compared to that emerged
in Williams et al. [
], maybe in relation to the different
techniques employed to analyse the microbiota.
Interestingly, when 4 or 8 month aged Ag ENM were used to
treat animals, microbiome alterations could not be
confirmed. These ENM, in fact, induced a less evident, if any,
inversion of the Firmicutes to Bacteroidetes ratio. Ag
ENM sulfidation, as a major transformation process for
ENM in contact with organic materials, was demonstrated
to be responsible for the reduction of aged Ag NP ENM
solubilization and Ag + ion release, that may all prevent
the gut microbiota alterations observed with freshly
prepared Ag ENM.
A polydisperse mixture of 60–100 nm Ag ENM (0–
100 μg Ag/kg for 4 h) incubated with ileal contents
sampled from weaned piglets, induced a dose-dependent
reduction in intestinal coliforms [
]. However, in the
same study, when pigs were treated with 20–40 mg Ag/Kg
for 2 weeks, only a non-significant trend toward coliform
reduction could be detected.
These results were in contrast with those obtained by
Hadrup et al. [
] in Wistar rats and Wilding et al. [
who found in C57BL/6NCrl mice that 28 days gavage
administration of 14–110 nm Ag ENM (2.25–10 mg/kg
bw/day) irrespective of their coatings, i.e. PVP or silver
acetate, did not affect the balance and number of the
two major bacterial phyla in the gut [
Interspecies differences in intestinal pH, gut microbiota,
diet as well as pathological conditions, which may affect
microbial composition generating significant inter-individual
variation, even in genetically identical animals with identical
starting microbial populations, may explain such different
outcomes. Certainly the ENM physico-chemical diversity,
in terms of size, coating, or other physicochemical
properties may have a different antimicrobial activity [
Additionally, the chemical transformations undergone by ENM
in aging consumer products as well as during digestion
processes may all affect the potential risk for microbial
alterations in real human conditions of exposure, particularly in
relation to the ENM solubilization ability. In this
perspective, to assess the degree, rate and duration of ion release
over time, also in in vitro models, should be verified as an
interesting instrument to predict the fresh or aged ENM
potential to affect microbial communities. Finally, the
experimental methodologies utilized in microbiota
investigations should be considered as a possible confounding issue
for the direct comparison of the data [
Sample type, collection site, the employment of a culture
strategy or not, lab techniques for the microbiota analysis
based on totally different approaches may all affect the final
outcomes of the studies and should be carefully considered
for an adequate interpretation of the results.
Effects detected by “omic” techniques
To gain insights into potential mechanisms of action of
ENM exposure on intestinal cells, biochemical changes
have been investigated by using “omics-” aproaches. By
using this technique, transcriptional effects involving an
enrichment of gene ontology categories related to
unfolded proteins, chaperons and stress responses were
detected after 5 μg/cm2 ZnO ENM exposure for 4 h of
Caco2 cells [
]. As far as epithelium permeability is
concerned, Brun et al. [
] demonstrated a significant
up-regulation in the expression of genes encoding
proteins involved in the maintenance of cell junctions in
Caco-2 and Caco-2-HT29-MTX models exposed to
50 μg/mL of TiO2 ENM for 6 h or 48 h; similar findings,
showing up-regulation of several genes involved into
tight junction and desmosome formation were reported
after exposure of T84 cells to Ag ENM (100 μg/mL for
48 h) [
]; by contrast a significant down-regulation of
genes encoding junctional proteins was observed by
Brun et al. [
] in the ileum of mice exposed to a single
gavage of 12.5 mg/kg TiO2 ENM, and sacrificed 6 h
after the gavage. These seemingly conflicting results
may at least in part be related to the different times
of exposure, which may allow, in the case of relatively
protracted exposure, the induction of compensative
mechanisms of repair.
In terms of nanosafety implications, genes whose
expression levels change significantly in a manner that
correlates with the effects of the ENM-exposure might be
useful as early nanotoxicity biomarkers.
As far as the mechanisms involved in the oxidative
stress are concerned, it has been reported the concomitant
down regulation of mammalian mitochondrial proteins,
and the up-regulation of those involved into the cellular
redox systems after exposure of LoVo cells to 10 μg/ml
for 24 h Ag ENM [
]. On the other hand, up-regulation
of cytosolic proteins associated with anti-oxidant activities
has been found, this finding being probably related to the
development of compensatory mechanisms [
Promising results have been obtained when using the
omics technique in order to discriminate between the
effects related to metallic ENM and those due to the release
of ions: as an example, a higher number of deregulated
proteins was detected after exposure to Ag NPs compared
to the ionic form [
Whether or not distinct pathways may be activated in
response to specific ENM has been investigated by Tilton
et al. [
], who performed global transcriptome and
proteome analyses of intestinal (Caco-2/HT29-MTX)
cocultured cells, exposed to 10 and 100 μg/ml TiO2
nanobelts (TiO2-NBs) and multi-walled carbon nanotubes
(MWCNT). Interestingly, the early 1 h post-exposure
transcriptional response was primarily independent of
ENM type, showing similar expression patterns in
response to both TiO2-NB and MWCNTs, while the 24 h
response was unique to each nanomaterial type. TiO2-NB
treatment affected several pathways, such as those
associated with inflammation, apoptosis, cell cycle arrest, DNA
replication stress and genomic instability, while MWCNTs
regulated pathways involved in cell proliferation, DNA
repair and anti-apoptosis.
Finally, the “omics” technique has also been exploited in
order to identify the mechanisms underlying the different
responses sometimes elicited by ENM of different size. It
has been recently reported that 20 nm sized Ag ENM
(1 μg/ml for 24 h) regulated different sets of proteins,
principally involved in pathogen-like response and in the
maintenance of the intestinal barrier function and
integrity, with a distinct pattern of cellular responses compared
to 200 nm Ag particles at the same experimental
conditions in a co-culture of Caco-2/HT29-MTX cells [
Impact of the inhaled enm on the git and occupational implications
GIT is a relevant target for extrapulmonary effects of
Inhalation is the main route through which people, in
particular workers, may come in contact with ENM, and
the lung is therefore the most obvious target of their
possible toxic effects. However, in recent years, a lot of
extrapulmonary effects of inhaled ENM, regarding
almost all organs and organ systems, have been reported
]. As summarized in Fig. 1, these effects may be
related to direct mechanisms (i.e. due to nanoparticles
crossing the alveolo-capillary barrier) or to indirect
mechanisms (i.e. due to the release of toxic mediators
following nanoparticles/lung interaction). It is important
to note that translocation to the systemic circulation is
very low, below 0.5% of the exposure concentration
], however, in the case of chronic exposure,
accumulation of nanoparticles in target organs might reach a
critical threshold causing injury.
GIT: An important overlooked target of extrapulmonary
effects of inhaled ENM
Among extrapulmonary effects, those on the
gastrointestinal tract have not explored yet. This is surprising,
because inhaled nanoparticles may reach the
gastrointestinal tract at a much larger amount than other
organs. In fact, like other organs and organ systems, the GI
tract can be exposed to nanoparticles crossing the alveolar
barrier and reaching the systemic circulation, as suggested
by the substantial fecal excretion of intravenously-injected
]. The amount of ENM reaching the gut through
the systemic circulation is probably greater than that
reaching other sites, as shown by Lee et al. who found that
silver ENM were transferred from systemic circulation
into the gut at a much higher rate than into the kidney or
other biological sites [
]. In addition to ENM crossing
the aveolar-capillary barrier (the only mechanism of direct
effect for other organs), the GIT may be also exposed a)
to inhaled ENM cleared from the lung through the
mucociliary escalator (which is a major clearance pathway for
ENM from the lung as compared with translocation
through the alveolo-capillary barrier [
] and b) to
nanoparticles directly ingested while breathing air (the so called
“aerophagia”). People affected by this common disorder
ingest air (and its content) at a much higher rate than
normal persons [
The relevance of gastro-intestinal exposure following
ENM inhalation is strongly supported by the recent
finding that after pulmonary exposure of rats to CeO2
ENM, the highest amount of ENM was recovered from
feces (71–90%), ENM recovered from the lungs being
7–18%, whereas urine and other extra-pulmonary organs
both contributed between 4 and 6% of the total
recovered mass [
]. Of note, the presence of ENM in feces
is by itself the proof of a significant interaction with the
GIT, since it implies a contact with the intestinal
microbiome/microbiota, a major player in GI physiology and
As reported for other organs and organ systems, there is
evidence that the gut may be sensitive to mediators
released by the inflamed lung, the so-called lung-gut axis.
This may be the case for interleukin-6 (IL-6), which is
systemically elevated in patients with emphysema [
is implicated in the pathogenesis of inflammatory bowel
]. In patients with asthma,
histopathological and functional alterations of the gastro-intestinal
tract have been described , probably related to the
circulation of activated lymphocytes between the mucosal
tissues of the lungs and of the gastrointestinal tract [
As shown in Fig. 1, systemic inflammation has been
reported after pulmonary exposure to ENM [
], and it is
considered a major pathophysiological mechanism in
order to explain the extrapulmonary effects of ENM. On
the basis of the above reported evidence, these effects are
to be expected also for the GI tract after inhalation of
In summary, on the basis of the currently available
evidence, not only the direct and the indirect mechanisms
evoked for the effects on other extrapulmonary sites are
plausible for the gastro-intestinal tract, but their impact
might be even greater for this biological site in comparison
Peculiar effects on GIT of inhaled ENM in comparison to
The reasons why the possible effects of inhaled ENM on
the GI tract have been neglected until now are probably
two: from one side the lack of substantial
epidemiological evidence of relevant gastrointestinal effects in
workers inhaling particles of larger size; from the other
one, the fact that the very large amounts of
nanoparticles ingested with food and drinks seem not to cause
As far as the first argument is concerned, only sparse
data linking exposure to particulate and functional [
or organic [
] GI diseases are available. Indeed, a
systematic investigation of this possible association has not
been performed. In any case, it should be considered that
ENM may have enhanced or novel toxic properties in
comparison to the same material in the bulk form,
therefore the lack of robust epidemiological data for the inhaled
bulk form cannot be translated to inhaled nanoparticles.
The assumption that ingested ENM are not harmful
(second point), is questioned by recent experiments
showing that ingested ENM may cause important
consequences on the homeostasis of the GI tract, in particular
on the gut microbiome, starting a chain of events
leading to significant physiological and anatomical
In addition, it should be considered that the biocorona
of inhaled nanoparticles is quite different in comparison
to that of ingested nanoparticles: the first are primarily
covered by biomolecules of the fluid lining the
respiratory tract, whereas the biocorona of the second ones is
mainly determined by the proteins, lipids and
carbohydrates present in the food, which they are usually
ingested with. The different biological identity between
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inhaled and ingested nanoparticles may be associated
with quite different biological effects, given the
increasing awareness of the role of biocorona in governing the
activity of nanoparticles in living organisms [
As an example, in experiments exploring the biological
fate of nanoparticles ingested with food, it was found
that gold nanoparticles ingested with milk are decorated
with beta lactoglobulin, a protein of bovine milk, and
that the protein is totally displaced by bile salts in the
small intestine (whose excretion from the gallbladder
into the intestine is in turn stimulated by food ingestion)
so that a complex formed by a core of gold nanoparticles
and a surface of bile salts is formed [
]. This complex
resembles the complex lipid droplet/bile salts, which
allows the absorption of lipids through the intestinal
epithelium, otherwise not permeable to them. It can
therefore be speculated that a similar phenomenon may
occur for inorganic nanoparticles ingested with milk,
allowing their transport through the intestinal epithelial
On the other hand, the bio-corona of inhaled
nanoparticles is characterized by a relatively fixed pattern of
phospholipids derived from the contact with the
pulmonary surfactant, whereas the protein composition
changes according to the surface properties of the
inhaled particles [
]. Nothing is known about the
interaction of this nanoparticle/biocorona complex with the
biological fluids of the gastrointestinal tract, (which are
in any case of different composition than those
encountered by nanoparticles ingested with food, due to the
lack of food-related stimulation of biliary and pancreatic
secretions) and we suggest that this topic should be
explored (see recommendations).
As highlighted above, pristine nanoparticles (i.e.
nanoparticles without a pre-formed biocorona) can also be
ingested with aerophagia. These nanoparticles are
covered in the stomach with a protein corona mainly
composed by pepsin, a proteolytic protein secreted by the
gastric chief cells. This protein seems to influence the
aggregation status of silver nanoparticles, which may
have implications on their toxicity [
the presence of a pepsin corona might explain the
reported lack of antimicrobial effect of silver nanoparticles
in the distal murine intestine [
A third type of biocorona may characterize ENM
reaching the GIT through the blood after pulmonary
pulmonary exposure: in this case ENM are covered with
a biocorona primarily formed in the lung and
subsequently modified in the blood [
]. Table 2 summarizes
the different biocorona composition of nanoparticles
reaching the gastro-intestinal tract through different
modalities, and highlights possible biological effects.
Another important point to be taken into
consideration is that nanoparticles reaching the gut following
inhalation may have a synthetic identity different from
that of ingested nanoparticles. As an example, some
nanoparticles at high risk of being inhaled (e.g. carbon
nanotubes) have a low chance to reach the gastrointestinal
tract through ingestion. Therefore, not only the same
nanoparticle may have different effects on the GI tract,
depending on the modality of exposure, but also some
nanoparticles reaching the gut following inhalation may
have a low chance to do so by ingestion with food and
may therefore cause biological responses which cannot be
observed with ingested nanoparticles.
The increasing interest in nanomaterials for advanced
technologies, consumer products, and biomedical applications
has led to great excitement about potential benefits, but
also concerns over the potential for adverse human health
effects. The gastrointestinal tract represents a likely route of
entry for many nanomaterials. In occupational settings,
gastrointestinal exposure may result from the mucociliary
clearance of inhaled nanomaterials, or from a direct
exposure in case of accidental events or when proper standards
of personal and industrial hygiene are not met [
The gastrointestinal epithelium and supporting
elements primarily act as a physical and biochemical barrier
between the luminal compartment and the interior of
the human body [
]. A key factor important to
understand the gastrointestinal toxicological profile of ENM is
the complex “interplay” between the great variability in
ENM physico-chemical properties and the absolutely
changeable conditions found along the gastrointestinal
system. ENM chemical composition,
structure/cristallinity, size and size distribution, shape, concentration,
surface area, functionality and charge may all vary
according to the methods of ENM production, preparation
processes, and storage, but may also be modified when
ENM are introduced into biological compartments. A
number of gastrointestinal luminal parameters, such as
physical forces, osmotic concentration, pH, digestive
enzymes, (i.e. buccal amylase, gastric pepsin, and intestinal
pancrease and lipase), together with different
gastrointestinal transit time, dietary status, other biochemicals and
commensal microbes may potentially impact ENM
properties therefore affecting their toxicological profile. In this
scenario, future researches should provide a systematic
and deeper characterization of both the primary
physicochemical features of ENM and those secondarily acquired
through the interactions occurring along the
gastrointestinal tract, e.g. the degree of aggregation or agglomeration
and the percentage of available ions for those ENM
undergoing dissolution, known influencing factors of NP
toxicity. Moreover, investigations focused on the possible
toxic impact of ENM on the gastrointestinal system
should elucidate which parameters are the strongest
inducers of any changes in ENM features, and, on the other
side, whether the full range of nanomaterials may be
modified in the gastrointestinal milieu, or if only certain
categories of ENM are subjected to such modifications [
to their intrinsically increased surface/mass ratio, ENM
may adsorb biomolecules on their surface upon contact
with food and/or biological fluids in gastrointestinal
compartments, resulting in the formation of a biomolecular
“corona” which may affect the uptake, bioaccumulation and
biotransformation of NPs possibly leading to unanticipated,
reduced or augmented, toxicities [
]. All these
aspects should be carefully considered to better correlate
ENM primary and acquired properties and biological
effects, in order to support the production of “ENM safe by
design” that, while maintaining most of the innovative and
revolutionary ENM features may, at the same time, be
characterized by lower toxicity .
Additionally, M cell- targeting of ENMs should be
carefully considered as another possible pathway of
interaction between ENMs and the intestinal milieu
which may have possible systemic implications. M cells
are specialized epithelial cells of the gut-associated
lymphoid tissues (GALT) that can play an
immunosensing and surveillance role by delivering luminal antigens
through the follicle-associated epithelium to the
underlying immune cells. Recent evidence has supported the
critical function of endogenous and synthetic
nanomineral chaperones in the efficient transport of molecules
across the epithelial barrier of the lymphoid follicles in
the small intestine [
]. In this perspective, further
investigation should be focused to assess whether ENMs
may be involved in protecting molecules from the GI
degradation, favoring an effective M-cell delivery, and a
greater transfection efficacy, therefore promoting
tolerogenic or stimulatory immunological responses. Overall,
this may be important to define the role of ENMs in
vaccine delivery systems for priming more effective humoral
and mucosal immune responses in the hosts .
A challenging issue relates also on the difficulties to
extrapolate experimental data to realistic
human/occupational exposure contexts. In vitro studies demonstrated
the ability of several types of ENM to induce cytotoxic,
inflammatory, oxidative stress as well as genotoxic responses
in exposed cells. However, in vitro models may not
accurately resemble the complexity of the in vivo response
]. Therefore, in the attempt to improve physiological
relevance of in vitro models and better mimic in vivo
gastrointestinal situations, including conditions of
inflamed mucosa, multi-cellular cultures have been
proposed. These may incorporate mucus secreting goblet
], microfold-cells [
], and even
immunecompetent macrophages and dendritic cells [
have shown a diverse, as well as more predictive of in vivo
response, susceptibility to the ENM injuries compared to
the cellular monolayers [
]. Moreover, for the assessment
of the toxicity of orally ingested ENM, additional
refinements, for instance, pre-treatment or co-administration of
particles with gastrointestinal reconstituted bio-fluids or
food matrix components, may be employed in order to
achieve more meaningful in vitro tests, with the aim to
deeply understand how protein corona changes may affect
ENM uptake, metabolism and toxicological behavior.
In vivo studies, on the other hand, can provide
information concerning ENM toxico-kinetics in gastrointestinal
and extra-intestinal tissues and ENM toxico-dynamic
behaviors in relation to their physicochemical properties. In
this regard, future in vivo studies should overcome the
difficulty to extrapolate findings from the generally,
higherdoses, short-term investigations on animal models, to real
low-dose, long-term conditions of exposure experienced
in general living and occupational settings, through the
adoption of more realistic experimental designs.
Moreover, in vivo studies should provide useful data to identify
possible biomarkers of exposure and early effect as well as
indicators of susceptibility to greater ENM induced
adverse health outcomes. Macrophage-mediated mucociliary
escalation followed by fecal excretion is a pathway for
clearing the inhaled NPs from the body [
Although it is rather difficult to routinely employ feces as a
suitable biological matrix for occupational biomonitoring,
on the account of the aforementioned clearance
mechanism, in the case of metal- or metal oxide-NP exposure, the
measurement of the elemental metal content in feces
should be viewed as a means to evaluate the recent/
current exposure to this kind of NPs . Moreover,
future investigations should explore possible biomarkers of
early effect, particularly as concerns mucosal
inflammatory alterations, which may be detected in fecal matrix.
Clinical experience, carried out with inflammatory bowel
diseases, Chron’s disease or ulcerative colitis, in this sense,
may provide useful suggestions for potential biomarkers
to be investigated and validated in the nano-toxicological
gastrointestinal field [
]. Additionally, taking advantage
of more innovative “omics techniques”, a comprehensive
analysis of differential gene and protein expression should
be performed to derive molecular profiles indicative of NP
exposure or early effect which may also explain possible
early modes of cellular response to NPs. This may be
helpful to understand also biological processes affected by
ENM or possibly involved in their toxico-dynamic
behavior to identify potential parameters of individual
susceptibility to ENM adverse effects [
]. Importantly, in the
attempt to define conditions of greater susceptibility to
ENM adverse outcomes, intra- and inter-individual
differences in normal physiology as well as in specific diseases
should be deeply analyzed. These conditions, in fact, may
alter the gastrointestinal environments affecting ENM
stability and movement as well as epithelial permeability.
Age, gender-specific differences, pregnancy status,
malnutrition, sleep cycle and stress, as well as inflammatory
bowel diseases can all result in increased lining
permeability and can augment the susceptibility to the absorption of
some types of ENM and to the induction of possible toxic
An emerging aspect that deserves deep attention
regards the potential interactions of ingested ENM with
the gut microbiota [
Few studies are still available concerning such
interesting issue, and some of them showed conflicting results.
In this field, some knowledge gaps should be overcome
by future investigations, particularly concerning which
pathological consequences may derive from microbiota
alterations induced by ENM. In an opposite perspective,
alterations in ENM toxico-kinetic and dynamic profile
caused by the same microbiota as well as by pre-existing
altered microbial states, such as gram negative bacterial
overgrowth, should be clarified. To deeply assess such
issues, fecal samples as representative models of the
microbiota of the colon, together with samples of the
human small intestine microbiota obtained from
ileostomies of people undergoing colon surgery, may be used.
Moreover, the employment of «humanized» models by
the inoculation of human gut microbiota to gnotobiotic
animals should be carefully considered as an ideal model
to study in vivo effects of ENM in order to transfer animal
data to humans. The study of the interactions between
ENM and the gastrointestinal tract may provide the
identification of innovative biomarkers based on the possible
specific modifications induced by ENM on the gut
microbiota. However, confounding effects related to individual
characteristics, pathological statuses, diet, drugs and
coexposure to other xenobiotics should be taken into careful
consideration to adequately interpret these results.
Overall, this information would provide deep insight
into possible ENM toxicological aspects that have not
been sufficiently explored up to date, with the aim to
reach a suitable assessment of risks in general living and
occupational ENM exposure settings.
In this perspective, another crucial aspect which
needs to be adequately explored in the future is
represented by the possible gastro-intestinal effects and
gastrointestinal-mediated systemic effects of inhaled
ENM. There is evidence that GIT may be a relevant
target for extra-pulmonary effects of inhaled ENM, and
that these effects may be different (and possibly more
relevant) than those induced by ingested ENM. In this
respect, experimental studies focused to this specific
topic are needed. We recommend in particular:
1. Assessing the impact of GI fluids and (gut)
microbiome on the biocorona of particles that are
deposited in the respiratory tract and after
mucociliary clearance being swallowed versus
nanoparticles ingested with food and how this
affects the biodistribution
2. Assessing the toxic effects of inhaled nanoparticles
(i.e. incubated with pulmonary surfactant) on gastric
cells, cells of the small intestine and cells of the colon
(including the interaction with the microbiome), as
compared with toxic effects of nanoparticles ingested
with food using in vitro methods
3. Perform a systematic comparison of effects of
inhaled nanoparticles on the gastrointestinal tract
and on intestinal microbiome compared with
The results of these studies might be the basis for
refining the focus on possible effects of ENM on human at
high risk of lung exposure (i.e. workers directly or
indirectly involved in nanotechnology).
The gastro-intestinal tract (GIT) is considered to be a
potential target of ENM ingested with food and water. It is
believed that the possible biological effects on the
gastrointestinal tract (GIT) deriving from ENM ingestion
involve mainly the consumers, whereas workers may be
only marginally affected, the inhalation being the main
way through which they may come in contact with ENM.
The biological effects of ENM on this organ are poorly
known both because of inherent difficulties in their
assessment due to the complex GIT environment and because
most available experimental studies suggest the lack of
In this review we discussed the most relevant gaps in
the knowledge of the biological effects of ENM on the
GIT and demonstrate that, by logically connecting the
available sparse information on this topic, it is possible to
identify sequential key processes, spanning from the
alterations of intestinal permeability to functional and organic
cellular damage, which may shed light on the
pathophysiological mechanisms of the gut/ENM interaction.
We also re-interpreted the results of some
experiments, such as, for example, the presence in the stools
of almost the total amount of ingested ENM, a finding
generally considered to be an indicator of the lack of
substantial local and systemic effects of the ingested
ENM; however, the recent evidence that ENM may have
a relevant impact on the gut microbiota, even in the
absence of substantial contact with GIT cells, indicates that
local and systemic biological effects mediated by changes
in gut microbiota are possible even in this situation.
Last but not least, we challenged the common belief
that the possible biological effects of ENM on the GIT
are confined to consumers, showing that inhaled ENM,
which represent the main route of ENM exposure for
workers, may induce peculiar and substantial effects on
the GIT: these effects may be different (and potentially
more important) than those related to ingested ENM.
Taken together, our findings suggest that the GIT
should have a primary role in the future research on the
biological effects of ENM. In this light, we identified and
suggested proper experimental protocols aimed to verify
Availability of data and materials
II and AP conceived the study, reviewed the literature, wrote and edited the
manuscript, provided overall guidance to the development of the manuscript;
EB critically reviewed and edited the manuscript; VL reviewed the literature and
wrote the manuscript; MC, LC, GDP, SI, AM, MM, PP critically reviewed the
manuscript and contributed to various sections; LP critically reviewed and
edited the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
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