J Occup Health 2009; 51: 64–73
Journal of
Occupational Health
Oral Lead Exposure Induces Dysbacteriosis in Rats
Rustam SADYKOV2, Ilya DIGEL1, Aysegül Temiz ARTMANN1, Dariusz PORST1, Peter LINDER1,
Peter KAYSER1, Gerhard ARTMANN1, Irina SAVITSKAYA2 and Azhar ZHUBANOVA2
1
Laboratory of Cellular Biophysics, Aachen University of Applied Sciences, Germany and 2Kazakh National
University Named after Al-Farabi, Republic of Kazakhstan
Abstract: Oral Lead Exposure Induces
Dysbacteriosis in Rats: Rustam S ADYKOV, et al .
Laboratory of Cellular Biophysics, Aachen
University of Applied Sciences, Germany—
Objectives: Lead’s (Pb(II)) possible role in intestinal
pathologies of microbial etiology remains mostly
unknown. The aim of this study was to examine the
effects of lead on the gut microbial community and its
interactions with rat intestinal epithelium. Methods:
The lead-induced changes in different intestinal
microbial groups (lactose-positive lac(+) and -negative
lac(–) E.coli strains, lactobacilli and yeasts) were
followed separately by the colony-forming unit (CFU)
method. Samples were taken from outbred white rats
subjected to different exposure schedules. Additionally,
the impact of different lead doses on microbial adhesion
to cultured intestinal cells (IEC-6) was investigated.
Finally, the lead accumulation and distribution were
measured by means of atomic absorption spectrometry.
Results: For the first time it was shown that oral lead
exposure causes drastic changes in the gut microbial
community. Proportional to the lead dose received,
the relative number of lactose-negative E.coli cells
increased dramatically (up to 1,000-fold) in comparison
to the other microbial groups during 2 wk of exposure.
Considering the number of microbes in the intestine,
such a shift in intestinal microflora (dysbacteriosis) is
very significant. Adhesion studies showed certain
stimulating effects of lead on E. coli attachment to rat
intestinal epithelium as compared to Lactobacillus
attachment. Conclusions: The mechanisms providing
the apparent competitive success of the lac(–) group
are unclear but could be related to changes in surface
interactions between microbial and host cells. This
study may provide important clues for understanding
the pathological effects of metal dietary toxins in human
beings.
Received Aug 4, 2008; Accepted Nov 7, 2008
Published online in J-STAGE Dec 19, 2008
Correspondence to: I. Digel, Laboratory of Cellular Biophysics,
Aachen University of Applied Sciences, FB9, Ginsterweg 1, 52428
Jülich, Germany (e-mail: [email protected])
(J Occup Health 2009; 51: 64–73)
Key words: Dysbacteriosis, IEC-6, Intestinal
microflora, Lead toxicity, Rat
Soluble lead, Pb (II), is one of the oldest known and
most studied occupational and environmental toxins.
Decades of use of lead-based paints and leaded gasoline
have resulted in widespread contamination. Although
lead has been later eliminated from household use, its
compounds are still widely used in industrial processes
and products1, 2).
Pb mainly affects the nervous system3), blood and
blood-forming organs, kidneys and gastrointestinal tract4).
There is no apparent threshold for lead toxicity 5),
suggesting that several different pathological mechanisms
are involved. The problem of lead presence in drinking
water6) and in airborne particles7, 8) remains acute in many
countries. Despite intensive study, there is still vigorous
debate about the toxic effects of lead, mainly concerning
low-level exposure in the general population owing to
environmental pollution by lead alone 9) and in
combination with other compounds10).
Total Pb dietary intake for an adult may vary from 20
to 150 µg per day in most countries11, 12) with a daily dose
of 0.2–2 mg regarded as safe13). The main routes of lead
entrance into the human body (food, water, dust) involve
the intestinal system. Young children are especially
susceptible to lead poisoning because they have 4–5 times
higher intestinal absorption. Lead absorption is
significantly influenced by the dietary intakes of iron,
calcium, phosphorus, pectin etc., and has been reviewed
extensively14–18). The most easily accessible forms of lead
are acetate, chloride and tetraethyl, whereas in the
intestine, lead is mainly bound to bile acids (cholic and
chenodeoxycholic) that contribute to lead transportation
(translocation) in the intestinal epithelium15).
Unabsorbed lead is excreted with feces.
The fact that intestinal epithelium and intestinal
microflora are usually subject to the highest Pb
Rustam SADYKOV, et al.: Lead-induced Dysbacteriosis in Rats
concentrations in the body implies the importance of the
detailed study of lead impact in this particular context.
The human gastrointestinal tract displays an enormous
variety of both aerobic and anaerobic microbial species.
Some 400–500 species belonging mainly to the
Escherichia, Bacteroides, Bifidobacterium, Lactobacillus
genera have been identified so far. While the contribution
of the indigenous gut microbial community is widely
recognized, only recently has there been evidence
pointing to indigenous flora in disease. Changes in
microbe-host relationships and following abnormal
expansion of gut microbial flora into the small bowel or
small intestinal bacterial overgrowth are known causes
of acute pancreatitis, bacterial gastroenteritis, irritable
bowel syndrome, and many other diseases19). F.Savino
has reported that changes in gut microflora could play an
important role in the pathogenesis of infantile colic, a
common problem in the first months of life20).
Some direct pathological effects of high doses of lead
on the intestinal system have been mentioned in the
literature and were attributed to dilation of the gastric
mucosal microcirculation accompanied by desquamation
of surface mucous cells21). In contrast, the influence of
sub-chronic intestinal lead exposure on the intestinal
microbial community and its relationships with intestinal
epithelium are still unestimated and poorly studied. This
is also true for many dietary toxins, for which the study
of the pathological effects is focused predominantly on
host tissues and cells, but totally neglects the responses
from commensal microbial flora.
The aim of the present study was to investigate the
influence of lead at different doses on the structure of the
rat intestinal microbial community in vivo and its
relationships to intestinal epithelium in vitro. The
understanding of the underlying role of altered
commensal gut microbiota in dietary toxicity could lead
to novel diagnostic and therapeutic strategies and would
contribute to public health.
Materials and Methods
Reagents
As a source of lead, soluble Pb salts (lead (II) acetate
and lead (II) chloride) were used. The lead concentrations
used in the experiments were calculated based on known
lead toxicity data. In the case of oral administration, the
50% lethal dose LD50 (rat) was assumed to be 4,665 mg/
kg for lead acetate Pb(CH3COO)2 (trihydrate form). The
toxicity data were obtained from accessible reference
books 5, 13) and material safety data sheets (MSDS)
provided by manufacturers22). All chemicals used were
reagent grade and purchased from Sigma-Aldrich Chemie
GmbH, Taufkirchen, Germany.
Animal experiments
White outbred adult male rats with a body mass of
65
180–220 g used for the experiments were obtained from
the Biology Faculty’s vivarium of Al-Farabi Kazakh
National University (Almaty). The experimental groups
and the concentrations of lead were chosen to give
maximum information from a reasonable number of
experiments. The rats were separated into one control
and 4 experimental groups, 3 rats in each group. All the
animals were maintained on a standard laboratory diet
(“Regal Rat” rat fodder), under the same standard
conditions. The “ad libitum” feeding regimen was used.
The first experimental group received 1/14 of oral LD50
(corresponding to 333.21 mg/kg body weight) every day
for 2 wk. The second, third and fourth experimental
groups received 1/60 (77.75 mg/kg), 1/240 (19.44 mg/
kg), and 1/960 (4.86 mg/kg), of LD50, respectively. The
lead acetate water solution (1 ml) was administered orally,
once a day, using a plastic syringe with a catheter. The
control group was administered 1 ml of sterile isotonic
NaCl solution.
Before the actual experiments began, the fecal samples
of all rats were collected for preliminary microbiological
and chemical analyses. Further samples were taken every
day during the whole period of lead administration (14
days). After 2 wk of lead exposure, the animals were
euthanized by carbon dioxide inhalation23). The whole
gastrointestinal tract from the lower esophagus to the
colon was removed. Blood and organs (large and small
intestine, stomach, kidneys and liver) were separately
analyzed for lead content using atomic absorption
spectrometry.
All animal procedures in this study were carried out
by licensed investigators and in accordance with regulated
procedures under the Animals (Scientific Procedures) Act
(1986).
Microbiological analysis
A common criterion for bacterial viability is the ability
to reproduce in a suitable nutrient medium (colony
forming unit (CFU) counting,). For analysis of microbial
flora present in excreta, samples of known mass
containing different concentrations of Pb were collected
individually from each rat (3 fecal samples from a single
rat, 3 rats in a group) and transferred under sterile
conditions into clean glass tubes. After that, the feces
were thoroughly homogenized in sterile phosphatebuffered saline (PBS; pH 7.4, 1:9, w/v) and serial 10 ×
dilutions were made to obtain 103, 10 5 and 107 times
diluted working solutions. From every dilution, 100 µl
were transferred into a Petri dish containing selective agar
culture medium and spread out by a glass spatula. The
media used were: Endo agar (for isolation and
identification of E.coli), MRS agar (for Lactobacillus sp.)
and Sabouraud agar (for yeasts). The media were
purchased from LabM company (International
Diagnostics Group, Lancashire, United Kingdom). The
66
Petri dishes containing MRS agar were placed into
specialized exsiccators to create anaerobic conditions.
After incubation at 37°C for 24–48 h, bacterial colonies
were counted (using a “blind” method to avoid subjective
bias in the results caused by experimenter’s intent) and
additionally identified using light microscopy and
corresponding microbiological staining. If the number
of colonies ranged between 10 and 300, the data were
included in the results. The successful counts of the
colonies were further calculated as concentrations of
colony-forming units per gram of fecal mass.
Atomic absorption spectrometry
The concentration of lead in organs and excreta was
determined by means of atomic absorption spectrometer
AAS-1N (Carl Zeiss Co, Jena, Germany) according to
the standard measurement protocol24). In brief, 500 mg
of homogenized whole organ was mixed in a quartz glass
with 25 ml of concentrated nitric acid and 25 ml of
double-distilled water, boiled, cooled down and mixed
with of 5% nitric acid to a final volume of 10 ml. The
sample was placed in the atomic absorption spectrometer
and ignited in propane-air flame. Then the lead-specific
283.3 nm absorption was measured and compared to
known standard values.
Adhesion assay
The bacterial strains used in the adhesion experiments
were Lactobacillus plantarum subsp. plantarum (ATCC
14917, serological group D), and Escherichia coli (ATCC
15223 serotype O13:H11, Lac– (i+ z+ y–)-mutable). The
microorganisms were cultured in culture media (MRS
and Trypton Soy Broth, respectively) to reach the
appropriate amount of biomass, washed 2 times with
sterile phosphate-buffered saline (PBS; pH 7.4) and kept
suspended in PBS until the experiments began.
As a model of intestinal epithelium, the normal rat
epithelial cells IEC-6 from the small intestine were used.
The IEC-6 cells were obtained from the German
Collection of Microorganisms and Cell Cultures (DSMZ).
They are adherent, transparent, epitheloid cells growing
as monolayers (polymorphy of cell forms). They
represent a non-continuous cell line with usually about
10–15 doublings and are able to synthesize fibronectin
and collagen25).
After thawing of the stock cells, the IEC-6 were
cultured in 45% Dulbecco’s medium (DMEM) containing
4.5 g/l glucose with addition of 45% RPMI 1640, 10%
fetal bovine serum and 0.1 U/ml insulin. To obtain the
necessary amount of biomass, the confluent cultures were
split from 1:2 to 1:4 about twice a week using trypsin/
EDTA, seeded out at approx. 3–6 × 105 cells/25 cm2 and
incubated at 37°C with 5% CO2.
For adhesion studies, the confluent monolayer of cells
(5th passage) was grown in 35 mm × 10 mm plastic
J Occup Health, Vol. 51, 2009
chambers with Nunclon Surface (Nunc GmbH,
Wiesbaden, Germany) in 2 ml of DMEM. Four-day postconfluent IEC-6 monolayers were washed twice with 1
ml of sterile DMEM (without phenol red indicator) before
the adhesion assay.
The amount of attached bacteria depended on their
initial concentration; therefore, the same final dilution of
bacterial suspension was used for all experimental groups,
allowing only the Pb concentration to vary between the
groups. Since the lactic acid bacteria have a tendency to
form chains and aggregates that make visual counting
difficult, in this study the microplate method was adopted
instead. After 24 h incubation of the monolayer with
culture medium containing different concentrations of
lead salts (both lead acetate and lead chloride were used),
the cells were washed twice with 2 ml of pure DMEM.
After that, 1 ml of the test bacteria suspensions at
concentrations between 105 and 108 CFU/ ml were added
to 1 ml of complete culture medium. This suspension (2
ml) was added to each chamber and incubated for 1 hour
at 37°C, in a 5% CO2 atmosphere, with gentle rocking26).
After that, the medium with non-adhered bacterial cells
was collected from each chamber into 3 microplate wells
(200 µl each) and the cell concentration was measured at
490 nm using a microplate reader (Bio-Rad Co. USA).
A lower optical density was interpreted as a lower
concentration of bacterial cell, and thus, as a case of better
adhesion.
For each concentration of lead, three plastic chambers
were used. Control groups (each in triplicate) were
created for adhesion experiments. In the first group, pure
medium without microbial cell was added to the
endothelial monolayer to check the contribution of the
adhered cells alone to the photometric signal. In the
second group, the microbial suspension was added into a
plastic chamber without endothelial cells to take into
account the unspecific adhesion of bacteria to plastic
surface. Finally, the optical density of the native microbial
suspension was measured to establish the initial reference
point.
At the end of the adhesion experiments, after the
samples had been taken for optical density measurements,
the monolayers were washed twice with sterile PBS (pH
7.4), fixed with methanol, Giemsa stained, and examined
microscopically27). Additional visual counting of adhered
cells was conducted in triplicate by two people and the
number of adherent bacteria was counted on about 1,000
IEC-6 cells, in 50 randomly selected microscopic fields.
Results
As shown in Fig. 1, even for the control group (left
raw) some amount of lead is present in rat organs, mainly
concentrated in the kidneys and large intestine, indicating
background lead consumption via drinking water, air dust
and fodder. The lowest dose of the lead used in these
Rustam SADYKOV, et al.: Lead-induced Dysbacteriosis in Rats
67
Fig. 1. Lead distribution measured by atomic absorption spectroscopy in rat
organs after 1 wk of oral lead intake. The concentrations are given as
fractions of LD50, which corresponds to 4.66 mg/g of body mass.
experiments (1/960 LD50 which corresponds to 4.86 mg/
kg), caused certain shifts in concentrations of lead in the
organs but did not visibly influence its distribution.
Further increases in the administered amount of lead
resulted in corresponding increases of its detectable
concentration in all the organs collected for measurement.
The collation of the data in Fig. 1 with the daily Pb
amount measured in rat feces during a 2-wk period of
time (Fig. 2 A,B) implies the constant increase of interintestinal Pb concentration even in the case of the lowest
dose given. Higher Pb concentrations caused
proportionally higher increases in the rate of Pb
accumulation without any visible saturation dynamics.
The changes observed in the relative amount of lactosepositive and lactose-negative E.coli cells as a reaction to
elevated Pb concentration (Fig. 3) were most interesting.
During the first 3–4 days of lead administration the
concentration of both E.coli lac(–) variants did not differ
between the control and the experimental groups.
However, the measurements made in the following 10–
12 days showed intensifying differences between lac(+)
and lac(–) E.coli responses to Pb presence. While the
lac(+) E. coli variants were, as expected, inhibited by the
presence of Pb in a concentration-dependent manner (Fig.
3 B), the lac(–) group appeared to use the weakening of
competition caused by Pb to its advantage in the same
concentration-dependent way. At the highest interintestinal Pb concentrations reached (10–15 mg/g feces,
see Fig.2), the dominance of lac(–) forms was obvious
(Figs. 3A and 4).
Figure 4 summarizes the changes induced in rat
intestinal microflora after 2 wk of continuous Pb (II)
intake according to the doses given. Though the absolute
and relative amounts of different intestinal bacteria in
the control group did not differ significantly after 2 wk
from the initial values (the two sets of bars on the left),
the smallest lead doses used in the experiment (4.86 mg/
kg and 19.44 mg/kg, corresponding to 1/960 of LD50 and
1/240 of LD50, respectively) resulted in distinct alterations
in bacterial microflora (Fig. 4, the two bar sets in the
middle).
To investigate possible reasons for such drastic changes
in the intestinal microbial community caused even by
low concentrations of lead, we performed a series of in
vitro experiments, using cultured rat intestinal cells, IEC6, and homogenous bacterial suspensions. After being
brought in contact with intestinal cell monolayer, some
of the applied bacteria firmly adhered to the cell surface.
High concentrations of lead acetate (1–5 mmole per
liter) showed in our studies distinct cytotoxic effects on
IEC-6 cells after 24 h incubation and, therefore, were
not used for adhesion studies. The dose of lead used for
adhesion experiments was in the range 0.1 nM–0.5 µM
per chamber (2 ml), which corresponds to 50 nM–0.25
mM concentrations in the culture medium. The adhesion
of the cells was measured spectrophotometrically which
was additionally controlled by microscopic observation
(Fig.5).
68
J Occup Health, Vol. 51, 2009
Fig. 2. A: Content of lead in rat feces measured during 2 wk of daily lead intake. B: Same data in logarithmic scale, to
show the lead accumulation in feces during the 2-wk period at low concentrations. The concentration of lead is
given as fractions of LD50, which corresponds to 4,665 µg of Pb acetate per kilogram of body mass.
Fig. 3. Dynamics of lac (–) (above) and lac (+) (below) E.coli in rat intestines in response to continual (2 wk) lead (II)
intake. The number of colony-forming units (CFUs) was counted every day for the different concentrations of Pb
administered and is shown respective to the dilution used. The number of CFU per gram corresponds to the
number of colonies (y-axis) × 104 for both lac(+)and lac(–) E.coli. The concentration of lead is given as fractions
of LD50, which corresponds to 4,665 mg/kg of rat body weight. *indicates the sets of data significantly (p<0.005)
different from control.
Rustam SADYKOV, et al.: Lead-induced Dysbacteriosis in Rats
69
Fig. 4. Changes induced in rat intestinal microflora after 2 wk of continuous Pb (II) intake
according to the dose given. The lactose-positive (+) and negative (–) E.coli colonies are
shown separately to show the impact of lead of lac-phenotype. The data is shown
irrespective of the suspension dilution used (104 E.coli lac(+), 104 E.coli lac(–), 107
Lactobacillus, 103 yeasts) to draw attention to the relative dynamics of the strains used. *
indicates the data are significantly (p<0.005) different from the control.
Fig. 5. Influence of lead on E.coli adhesion to rat intestinal cells, IEC-6. A: Control flask. B: The
dose of lead chloride is 0.05 µM per chamber (2 ml). C: The dose of lead chloride is 1 µM
per chamber. Black dots (indicated by arrows) are bacterial cells firmly attached to the
epithelial monolayer.
It was found that for lactose-negative E.coli cells, a
broad interval of lead salt concentrations exists (2.5–250
µM), where the adhesion of the bacteria to intestinal cells
was stimulated compared to the control. The maximum
of E.coli adsorption (approx. 104–108% relative to the
control) was observed at –78 µM concentration (for both
salts used) corresponding to the dose –0.1565 µmol per
flask (Fig. 6). In Fig. 6B the points situated above the x-
axis can be interpreted as decrease in attachment and those
lying below as increase in attachment. In contrast to E.
coli, adhesion of Lactobacillus cells was significantly
inhibited by lead, showing some increase at only one
concentration (–326 µ M) which could possibly be
attributable to electrostatic interactions. Similar effects
were also found when lead (II) nitrate was used as a source
of lead but the contribution of the nitric group cannot be
70
J Occup Health, Vol. 51, 2009
Fig. 6. Comparison of E.coli (lac-) and Lactobacillus plantarum adhesion to rat intestinal
cells according to lead acetate dose. A: Standard scale. B: The same data shown in
logarithmic scale. The stimulation of adsorption of lactose-negative E.coli cells by
Pb(II) is visible in data points below the x-axis.
excluded in this case.
Discussion
A great amount of research effort has been devoted to
the description of intestinal adsorption of metals28) but
their effects on intestinal microflora have not drawn any
serious attention. Previous studies have suggested that
lead toxicity is the result of its effect on calcium
metabolism17), and channel dysfunction29). Nevertheless,
some mechanisms underlying lead toxicity at low
concentrations are still unclear, maybe because of the
absence of a systemic approach to understanding lead
toxicity. Recent studies have revealed the intricate
relationship between the vast population of microbes that
live in our gut and the human host30). Our experiments
were designed, first, to investigate the effects of lead on
the rat intestinal microbial community, and second, to
study if the effect is related to microbial adhesion to rat
intestinal epithelial cells.
The rat, as well as its intestinal cells, has been shown
to be a good model for toxicological animal-human
bioavailability correlations 16, 31–33). Many aspects of
concentration and distribution of lead in rat resemble
those in the human body. Nevertheless, some interspecies
variations in intestinal absorption (0.36 ± 0.07 for human
beings and 0.23 ± 0.02 for rats) should be taken into
account.
The results of our atomic absorption spectroscopy
measurements of lead distribution in rat organs were in a
good accordance with data published previously34). They
show in these experiments the lead distribution
corresponded to typical profiles and was not influenced
by unaccounted factors35). Interestingly, the higher doses
of Pb caused the accumulation of Pb mainly in the
gastrointestinal system (small and large intestine and
stomach) indicating the growing inability of intestinal
epithelial transport mechanisms to absorb Pb.
The next step was to study the dynamics of the lead
concentration in intestinal contents and feces during 2
wk of chronic exposure to evaluate the capacity of
intestinal absorption processes. Risk assessments for
toxicants in environmental media via oral exposure often
rely on measurements of total concentration in a collected
sample. However, the digestive system cannot mobilize
all of a toxicant present in the binding matrix, and cannot
absorb it with near 100% efficiency. Default values of
30% lead bioavailability are assumed to be a reasonable
indicator for dose, but the recent studies by Yu and
Rustam SADYKOV, et al.: Lead-induced Dysbacteriosis in Rats
coauthors suggest that higher values for gastric
bioaccessibility of lead possibly need to be considered36).
Our measurements revealed that the lead concentration
in feces correlated well with the dose administered. Even
in the case of small concentrations, the lead content
gradually increased during the experiment, indicating a
decreasing trend for lead absorption by gut epithelium,
resulting in accumulation in the intestinal space. As a
result, intestinal microorganisms were exposed to
continuously increasing concentrations of lead. Taking
into account that different microbial groups have different
susceptibilities to lead, population shifts in intestinal flora
were to be expected. Indeed, the CFU method showed
clearly that the intake of lead (II) caused explosive growth
of lactose-negative E.coli variants. In contrast, the yeasts,
lactobacilli and lactose-positive coli bacteria were
suppressed in proportion to lead concentration. Although
particular health-related consequences of the shift in
proportions of non-lactose fermenting relative to lactosefermenting bacteria are not clear yet, rapid uncontrollable
proliferation of lac(–) strains could be considered as an
ecological imbalance causing a pathological state, referred
to as dysbacteriosis, which affects many aspects of
gastrointestinal system function.
One important aspect yet to be studied is to what extent
the lac(–) strains differ from lac(+) strains. Sometimes
E.coli strains can be lac(–) on first isolation and become
lac(+) after subculture.
The gastrointestinal microflora is a complex ecological
system, normally characterized by a flexible equilibrium.
Bifidobacteria and lactobacilli are Gram-positive lactic
acid-producing bacteria constituting a major part of the
intestinal microflora in humans and other mammals37).
Successful probiotic bacteria are usually able to colonize
the intestine, at least temporarily, by adhering to the
intestinal mucosa. By doing that, probiotic bacteria might
prevent the attachment of pathogens, such as coli-form
bacteria and clostridia, and stimulate their removal from
an infected intestinal tract. Administration of
antimicrobial agents notoriously causes disturbances in
the ecological balance of the gastrointestinal microflora
with several unwanted effects such as colonization by
potential pathogens38), many of them belonging to lactosenegative coli bacteria39, 40). To our knowledge, our study
is the first one to show a relation between dysbacteriosis
and oral lead exposure.
The most important role of the commensal microflora,
from the point of view of the host, is to act in colonization
resistance against exogenous, potentially pathogenic,
microorganisms 41). Thus, one of the causes of the
observed lead-induced dysbacteriosis could be the
disturbance in microbial adhesion to epithelia. To verify
this hypothesis, a series of model in-vitro experiments
can be performed. Recently, several laboratory models
using human and rat intestinal cell lines have been
71
developed to study the adhesion of probiotic lactic acid
bacteria42–44). The study by Dekaney and co-workers42)
showed the relevance of use of cultured rat intestinal
epithelial cells (IECs) as a model for studying Pb transport
as well as Pb accumulation. They found that Pb uptake
by IECs depended on the extracellular Pb concentration.
In our experiments, cultured intestinal cells IEC-6 were
first exposed to lead, and then their ability to bind different
bacteria was examined.
One of the principal difficulties in modeling intestinal
environments lies in the uncertainty over mechanical
stresses, and the topology and composition of the surfaces
involved. The cultured cell model is a simple system
that excludes many physiological variables such as
peristalsis, blood flow, gastric emptying, etc., allowing
the adsorption processes to be studied in isolation43, 44).
Nevertheless, there are obvious limitations with cell
culture techniques, including the lack of systemic control,
short incubation times, etc. Thus, we realize that in these
studies the adhesion process only partially mimics the in
vivo interactions of bacterial and intestinal cells and that
the data must be interpreted guardedly.
Our in vitro study showed clear differences in lead
influence on adsorption profiles between Lac– (i+ z+ y–)
E. coli strain (representing a pathological group) and
Lactobacillus plantarum ATCC 14917 (known as a
probiotic strain). In contrast to Lactobacillus, there was
a broad range of lead concentrations where the adhesion
of E.coli cells was not inhibited but rather stimulated as
compared to the control. The particular molecular reasons
for this phenomenon are unclear and could include both
unspecific changes in electrostatic interactions and Pbinduced conformational rearrangements of surface
proteins.
Conclusions
Several conclusions can be drawn. First, the oral
exposure of human beings and animals to low Pb
concentrations deserves more careful attention. Lead
doses that were much lower than those causing visible
harmful effects on cell culture or on the level of the whole
body could induce significant changes in intestinal
microflora. The presence of Pb somehow creates selective
advantages for pathological lactose-negative coli forms
and thus contributes to dysbacteriosis development.
Second, the adhesion of bacteria to intestinal epithelium
is a possible cause of lead-induced dysbacteriosis. Third,
the results could be possibly extrapolated to human
beings, opening the opportunity of understanding some
mysterious cases of persistent dysbacteriosis as a result
of latent Pb intoxication. This, in turn, would suggest
probiotic treatment as a promising therapy for leadexposed patients on the one hand and the possibility of
Pb-scavenging therapy for some patients with
dysbacteriosis on the other.
72
Regarding public health, monitoring and control over
Pb intestinal levels would not only diminish the risk of
lead specific pathologies, but would also be of major
importance in maintaining the normal gut microflora.
Acknowledgments: The study was supported by PhD
Program of Kazakh National University and Institut für
Bioengineering in Jülich. The authors thank Baurzhan
for invaluable help in experiments.
J Occup Health, Vol. 51, 2009
15)
16)
17)
References
1) Mahaffey KR. Environmental lead toxicity: nutrition
as a component of intervention. Environ Health
Perspect 1990; 89: 75–8.
2) Elinder CG, Lind B, Nilsson B, Oskarsson A. Wine—
an important source of lead exposure. Food Addit
Contam 1988; 4: 641–4.
3) Chang LW. The neurotoxicology and pathology of
organomercury, organolead, and organotin. J Toxicol
Sci 1990; 4: 125–51.
4) Gerhardsson L, Hagmar L, Rylander L, Skerfving S.
Mortality and cancer incidence among secondary lead
smelter workers. Occup Environ Med 1995; 10: 667–
72.
5) Flury F, Zernik F. Zusammenstellung der toxischen und
letalen dosen für die gebräuchlichsten gifte und
versuchstiere. Abder Hand Biol Arbeitsmethod 1935;
4: 1289–422.
6) Maas RP, Patch SC, Morgan DM, Pandolfo TJ.
Reducing lead exposure from drinking water: recent
history and current status. Public Health Rep 2005; 3:
316–21.
7) Firestone M, Sonawane B, Barone S Jr, Salmon AG,
Brown JP, Hattis D, Woodruff T. Potential new
approaches for children’s inhalation risk assessment. J
Toxicol Environ Health A 2008; 3: 208–17.
8) Chan LY, Kwok WS, Chan CY. Human exposure to
respirable suspended particulate and airborne lead in
different roadside microenvironments. Chemosphere
2000; 1–2: 93–9.
9) Cunningham G. Lead—toxicology and assessment in
general practice. Aust Fam Physician 2007; 12: 1011–
13.
10) Lukacs A, Lengyel Z, Institoris L, Szabo A. Subchronic
heavy metal and alcohol treatment in rats: changes in
the somatosensory evoked cortical activity. Acta Biol
Hung 2007; 3: 259–67.
11) Sharma RK, Agrawal M. Biological effects of heavy
metals: an overview. J Environ Biol 2005; 2 (Suppl):
301–13.
12) Tahiri M, Pellerin P, Tressol JC, et al. The
rhamnogalacturonan-II dimer decreases intestinal
absorption and tissue accumulation of lead in rats. J
Nutr 2000; 2: 249–53.
13) Anderson LA Jr. Defining the lead detection and
abatement industry: a review of the AIHA white paper
on environmental lead. American Industrial Hygiene
Association. Am Ind Hyg Assoc J 1994; 4: 300–3.
14) Blake KC, Mann M. Effect of calcium and phosphorus
18)
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)
29)
30)
31)
32)
on the gastrointestinal absorption of 203Pb in man.
Environ Res 1983; 1: 188–94.
Conrad ME, Barton JC. Factors affecting the absorption
and excretion of lead in the rat. Gastroenterology 1978;
4: 731–40.
Khotimchenko M, Serguschenko I, Khotimchenko Y.
Lead absorption and excretion in rats given insoluble
salts of pectin and alginate. Int J Toxicol 2006; 3: 195–
203.
Kim KA, Chakraborti T, Goldstein GW, Bressler JP.
Immediate early gene expression in PC12 cells exposed
to lead: requirement for protein kinase C. J Neurochem
2000; 3: 1140–6.
Ziegler EE, Edwards BB, Jensen RL, Mahaffey KR,
Fomon SJ. Absorption and retention of lead by infants.
Pediatr Res 1978; 1: 29–34.
Schmidt C, Stallmach A. Etiology and pathogenesis of
inflammatory bowel disease. Minerva Gastroenterol
Dietol 2005; 2: 127–45.
Savino F, Cresi F, Pautasso S, et al. Intestinal microflora
in breastfed colicky and non-colicky infants. Acta
Paediatr 2004; 6: 825–9.
Verschoyle RD, Little RA. The acute toxicity of some
organolead and organotin compounds in the rat, with
particular reference to a gastric lesion. J Appl Toxicol
1981; 5: 247–55.
Sigma-Aldrich Co. Lead acetate trihydrate ACS
reagent. 2007; 215902.
Pierce S. Eutanasia of Mice and Rats. Internet Link
2006.
Yee HY, Nelson JD, Jackson B. Measurement of lead
in blood by graphite furnace atomic absorption
spectrometry. J Anal Toxicol 1994; 7: 415–8.
Quaroni A, Wands J, Trelstad RL, Isselbacher KJ.
Epithelioid cell cultures from rat small intestine.
Characterization by morphologic and immunologic
criteria. J Cell Biol 1979; 2: 248–65.
Lee YK, Lim CY, Teng WL, Ouwehand AC, Tuomola
EM, Salminen S. Quantitative approach in the study
of adhesion of lactic acid bacteria to intestinal cells
and their competition with enterobacteria. Appl Environ
Microbiol 2000; 9: 3692–7.
Mange JP, Stephan R, Borel N, et al. Adhesive
properties of Enterobacter sakazakii to human epithelial
and brain microvascular endothelial cells. BMC
Microbiol 2006; 58–67.
Whitehead MW, Farrar G, Christie GL, Blair JA,
Thompson RP, Powell JJ. Mechanisms of aluminum
absorption in rats. Am J Clin Nutr 1997; 5: 1446–52.
Gavazzo P, Zanardi I, Baranowska-Bosiacka I,
Marchetti C. Molecular determinants of Pb(2+)
interaction with NMDA receptor channels. Neurochem
Int 2008; 1–2: 329–37.
Othman M, Aguero R, Lin HC. Alterations in intestinal
microbial flora and human disease. Curr Opin
Gastroenterol 2008; 1: 11–6.
Crouthamel WG, Abolin CR, Hsieh J, Lim JK. Intestinal
pH as a factor in selection of animal models for
bioavailability testing. J Pharm Sci 1975; 10: 1726–7.
Iturri SJ, Pena A. Heavy metal-induced inhibition of
Rustam SADYKOV, et al.: Lead-induced Dysbacteriosis in Rats
33)
34)
35)
36)
37)
38)
active transport in the rat small intestine in vitro.
Interaction with other ions. Comp Biochem Physiol C
1986; 2: 363–8.
To m c z o k J , G r z y b e k H , S l i w a W, P a n z B .
Ultrastructural aspects of the small intestinal lead
toxicology. Part I: surface ultrastructure of the small
intestine mucosa in rats with lead acetate poisoning.
Exp Pathol 1988; 1: 49–55.
G r i e c o B , P e n n a r o l a R , L a m a n n a P.
Histoautoradiographic study of the distribution of
radioactive lead (Pb 21O) in various rat organs. Folia
Med (Napoli) 1966; 12: 937–47.
Yoshikawa H, Suzuki, Y. Effect of phenobarbital on
the distribution and excretion of lead in rats acutely
poisoned with lead. Toxicol Lett 1981; 1: 51–4.
Yu CH, Yiin LM, Lioy PJ. The bioaccessibility of lead
(Pb) from vacuumed house dust on carpets in urban
residences. Risk Anal 2006; 1: 125–34.
Jonkers D, Stockbrugger R. Review article: probiotics
in gastrointestinal and liver diseases. Aliment
Pharmacol Ther 2007; 26 (Supple 2): 133–48.
Gus’kova T, Pushkina T, Radkevich T. Possibility of
dysbacteriosis development upon using drugs
73
39)
40)
41)
42)
43)
44)
belonging to different pharmacological groups. Pharm
Chem J 2006; 7: 10–1.
Collado MC, Jalonen L, Meriluoto J, Salminen S.
Protection mechanism of probiotic combination against
human pathogens: in vitro adhesion to human intestinal
mucus. Asia Pac J Clin Nutr 2006; 4: 570–5.
Salminen S, Benno Y, de Vos W. Intestinal colonisation,
microbiota and future probiotics? Asia Pac J Clin Nutr
2006; 4: 558–62.
Ouwehand AC, Niemi P, Salminen SJ. The normal
faecal microflora does not affect the adhesion of
probiotic bacteria in vitro. FEMS Microbiol Lett 1999;
1: 35–8.
Dekaney CM, Harris ED, Bratton GR, Jaeger LA. Lead
transport in IEC-6 intestinal epithelial cells. Biol Trace
Elem Res 1997; 1–2: 13–24.
Greene JD, Klaenhammer TR. Factors involved in
adherence of lactobacilli to human Caco-2 cells. Appl
Environ Microbiol 1994; 12: 4487–94.
Tuomola EM, Salminen SJ. Adhesion of some probiotic
and dairy Lactobacillus strains to Caco-2 cell cultures.
Int J Food Microbiol 1998; 1: 45–51.