Heavy metals are biologically nondegradable environmental pollutants that can negatively affect the health of human and animals prompting carcinomas, hematotoxicity, allergy and immunotoxicity (Järup, 2003). They accumulate within the food chain what results in serious ecological and health hazards. Exposure of organisms to the heavy metal ions impairs hosts immunocompetence and increases susceptibility to the infections. Heavy metals also affect the cell physiology and modulate immune system responses (Valko
Due to high abundance of industrial heavy metals in exposed areas small mammals migrating in affected territories are suitable bioindicators of anthropogenic environmental pollution. Survival of the small mammals allows identify local environmental problems such as long-term effect of heavy metals bioaccumulation and determine health defects caused by ongoing environmental pollution (Flickinger & Nichols, 1990; Tersago
Environmental pollution can affect parasitism (Lafferty & Kuris, 1999) where parasites can be used as sentinel organisms i.e. indicators of environmental pollutants accumulation. On the other hand parasites can interfere with bioindicative potential of hosts due to their effect on physiology and behaviour. It may trigger a false positive or negative assessment of the environmental contamination. Therefore it is required to study the parasite-host interactions in relation to the heavy metal immunotoxicity.
The immunotoxicity of heavy metals and their modulation of parasite-host interactions were tested on model of
The aim of this study was to examine the immunotoxic effect of heavy metals (lead, cadmium and mercury) on cytokine production and development of the Th1/Th2 type of response during subsequent murine
Mice were exposed to the salts of lead or cadmium dissolved in drinking water and mercury dissolved in
Cadmium: CdCl2 (Sigma-Aldrich, Hamburg, Germany) 100 mg/l, provided in drinking water
Mercury: HgCl2 (Sigma-Aldrich, Hamburg, Germany) 0.2 mg/kg of body weight, injected subcutaneously (s.c.) at each day throughout the experiment.
The embryonated
Each mouse received a dose of 1000 infective
Three experiments were carried out on pathogen-free 8 weeks old male BALB/c mice (VELAZ, Prague, Czech Republic; n=180) weighting 18 – 20 g. Mice were kept in controlled environment under a 12-h light/dark regimen at room temperature (22 – 24 °C) with 56 % humidity and fed with commercial diet (MP-OŠ-06, MIŠKO, Snina, Slovak Republic). Water was provided ad libitum. The experimental protocol complied with current Slovak ethics law and was approved by the Animal Care Committee of the Institute of Parasitology SAS and the State Veterinary and Food Administration of the Slovak Republic (No. Ro-1888/10-221a). Three individual experiments with heavy metals intoxicated mice were performed.
Experiment I. – Chronic intoxication with lead (Pb) + infection
Experiment II. – Chronic intoxication with cadmium (Cd) + infection
Experiment III. – Chronic intoxication with mercury (Hg) + infection
Animals in each independent experiment were divided randomly
into four groups as follows:
Group 1 (n=18) – control mice without intoxication and infection.
Group 2 (n=18) – mice intoxicated with Pb or Cd or Hg.
Group 3 (n=12) – mice infected with 1000 eggs of
Group 4 (n=12) – mice intoxicated with Pb or Cd or Hg and subsequently infected with
Samples of the spleen, liver, lungs, kidneys and muscles were obtained on days 0, 7, 14, 21, 25 (infected groups 3, 4), 28 and 35. Three mice were examined on each sample day and from each experimental group.
Murine tissue samples (liver, kidneys, muscle) for detection of lead and cadmium content were mineralized with HNO3 and H2O2 in Microwave oven MLS 1200 (Milestone, Sorisole, Italy). The concentrations of Pb and Cd were quantified by Inductive Couple Plasma-OES (Perkin Elmer Instrument USA, Optima 200 DV, Richmond, California) using the wavelength 220.353 and 228.802 nm for Pb and Cd, respectively. The concentration of mercury was determined directly by a single-purpose atomic absorption spectrometer AMA 254 (Advanced Mercury Analyser, Altec, Prague, Czech Republic).
Surgically removed livers or lungs were cut into small pieces. Then packed in sterile gauze and placed at the top of a conic tube filled with warm phosphate buffered saline (PBS). Living larvae were recovered from tissues in Baermann’s apparatus after overnight incubation at 37 °C. The larval suspensions were centrifuged at 200
The capture ELISA was employed to determine the concentration of
Statistical differences were assessed by Two-way ANOVA followed by Bonferroni post-tests using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA, www. graphpad.com). Values of P<0.05 were considered significant what allowed comparison between each experimental groups and at each time point (control versus heavy metal without parasitic infection and
The heavy metal accumulation was determined in the liver, kidneys and muscles of intoxicated mice as well as in mice intoxicated and subsequently infected with
Heavy metal concentrations in tissues of mice intoxicated with heavy metals (lead Pb, cadmium Cd, mercury Hg) and infected with
Heavy metal | Experimental groups | Days of experiment (Days post infection) | liver (mg/kg) (mean±S.D.) | kidneys (mg/kg) (mean±S.D.) | Muscles (mg/kg) (mean±S.D.) |
---|---|---|---|---|---|
Pb | control | 0 | 0.12 ± 0.06 | 0.22 ± 0.08 | 0.16 ± 0.09 |
intoxication (before infection) | 21 | 0.51 ± 0.18 | (P<0.05; | 0.71 ± 0.18 | |
intoxicated | 35 | 0.27 ± 0.03 | 0.34 ± 0.04 | 0.48 ± 0.04 | |
intoxicated+infected | 35 (14 p.i.) | 0.36 ± 0.14 | 0.44 ± 0.02 | 0.59 ± 0.14 | |
Cd | control | 0 | 0.02 ± 0.01 | 0.05 ± 0.01 | 0.03 ± 0.01 |
intoxication (before infection) | 21 | (P<0.01); | (P<0.01); | 0.04 ± 0.01 | |
intoxicated | 35 | (P<0.05; | (P<0.0001) statistically significant differences between control and heavy metal’s intoxication; | 0.03 ± 0.01 | |
intoxicated+infected | 35 (14 p.i.) | 0.35 ± 0.30 | (P<0.05) statistically significant differences between heavy metal’s intoxication and heavy metal’s intoxication in combination with | 0.05 ± 0.01 | |
Hg | control | 0 | 1.03 ± 0.35 | 0.98 ± 0.39 | 0.03 ± 0.01 |
intoxication (before infection) | 21 | (P<0.0001) statistically significant differences between control and heavy metal’s intoxication; | (P<0.0001) statistically significant differences between control and heavy metal’s intoxication; | (P<0.01); | |
intoxicated | 35 | (P<0.0001) statistically significant differences between control and heavy metal’s intoxication; | (P<0.0001) statistically significant differences between control and heavy metal’s intoxication; | (P<0.01); | |
intoxicated+infected | 35 (14 p.i.) | 9.57 ± 4.12 | 90.68±1.57 | 0.52±0.18 |
In comparison with the only infected mice without heavy metals intoxication (Table 2) the larval recoveries from the liver and lungs were increased in mice intoxicated with Pb. However, no significant differences were found. Mice intoxicated with Pb showed slight weight loss (unpublished data).
Parasite larval burden in mice intoxicated with heavy metals (lead Pb, cadmium Cd, mercury Hg) and infected with
Heavy Metal Intoxication | Days of experiment (Days post infection) | intoxication + | |||
---|---|---|---|---|---|
Liver (mean±S.D.) | lungs (mean±S.D.) | lungs (mean±S.D.) | lungs (mean±S.D.) | ||
Pb | 25. (4. p.i.) | 195.0 ± 43.0 | 250.6 ± 28.7 | ||
28. (7. p.i.) | 3.3 ± 1.5 | 66.7 ± 15.9 | 2.6 ± 3.8 | 86.3 ± 17.5 | |
Cd | 25. (4. p.i.) | 225.5 ± 57.3 | P<0.05) statistically significant differences between | ||
28. (7. p.i.) | 7.3 ± 3.5 | 31.7 ± 23.6 | 2.3 ± 4.0 | 33.3 ± 2.1 | |
Hg | 25. (4. p.i.) | 179.3 ± 40.5 | P<0.05) statistically significant differences between | ||
28. (7. p.i.) | 4.3 ± 3.2 | 25.7 ± 6.1 | 4.3 ± 4.2 | P<0.05) statistically significant differences between |
The production of IL-5 by splenocytes (Fig. 1a) was stimulated during Pb intoxication after 3 weeks of administration (P<0.05).
The TNF-α and IFN-γ production (Fig. 1c, d) was suppressed from week 2 of Pb intoxication (P<0.01) until the end of the experiment (3 and 4 weeks, P<0.0001; and 5 weeks, P<0.001).
The total number of
The IL-5 production (Fig. 2a) was not markedly influenced by Cd whereas
TNF-α synthesis was increased by Cd intoxication (P<0.01; P<00.05; P<0.001) throughout the experiment (Fig. 2c).
The total numbers of larvae in mice intoxicated with Hg (Table 2) were significantly reduced in the liver at day 4 p.i. and in the lungs at day 7 p.i. Mice intoxicated with Hg were extremely cachectic (unpublished data).
The IL-5 production was elevated (P<0.0001) at the 3rd week of Hg intoxication and
Hg intoxication increased the TNF-α production (P<0.001; P<0.0001) from week 3 until the end of the experiment (Fig. 3c).
Ability to accumulate heavy metals in tissues is associated with modulation of the host immune response what can explain positive or negative role of contaminated environment on the outcome of parasitic infection. The consequence of this interaction on small mammals living in terrestrial ecosystems has great bioindicative value for the assessment of environmental contamination. Current immunological analysis about the effect of heavy metals in model parasitic zoonosis is pilot study examining parasite-host interactions under heavy metal stimulus. Under such conditions, both the host and parasite are susceptible to the pathogenic effects of toxicants which may result in detrimental changes to their immunological and physiological responses. To this point it is not known how combination of contaminated environment and parasitism can influence development of immune response where regulatory (helper lymphocytes, cytokines) and effector (macrophages) components are important in the host defence against parasite. Specifically when these immune cells are also target structures for the heavy metals (Šoltys
Mice are monogastric animals in which the liver and kidneys serve as detoxification organs that help to eliminate toxic waste metabolites from the body. Heavy metals can enter the host organism either through the plant food or water contaminated with toxic substances from nearby industrial complexes (Hančuľák
We found that the differences in parasite infection intensity were dependent on particular heavy metal involved. In summary a non-significant increase parasite burden in mice intoxicated with Pb was recorded. In contrast to this observation a big parasite reduction in Cd intoxicated mice was observed. Also a low numbers of Ascaris larvae were found in Hg intoxicated mice. Moreover these animals were extremely cachectic and probably their extreme weakness limited the development of parasitic infection. The differences in parasite burden may well be explained by a different immunotoxic effect of heavy metals on the host cytokine responses. Lead intoxication lead to the development of Th2 type of response where, to some extent, an increase in production of anti-inflammatory cytokine IL-10 and IL-5 from week 2 was observed. The Th1 response was suppressed after the Pb administration. IFN-γ and TNF-α concentrations were greatly below the control levels. Our data correlates with results of Hemdan
Mice mercury intoxication did not show a clear cut between Th1 or Th2 type of response. Mercury significantly stimulated the production of IL-10 what also affects the synthesis of Th1 cytokines. Correspondingly, the other authors confirmed that Hg affects immune function in human cells by dysregulation of cytokine signalling pathways. Gardner
In our experiment mice intoxicated with heavy metals and subsequently infected with
The Th1 immune response is effective in eliminating of tissue stages of these parasites (Mulcahy
Only cadmium intoxication stimulated the IFN-γ production and subsequent parasite infection did not change significantly the IFN-γ level. Up-regulation in TNF-α induced by cadmium was turned over into suppression after the infection. Reduced number of
The protective immune response against the gastrointestinal helminths is characterized by Th2 response. Individuals that can naturally upregulate the Th2 response during larval migration may cause suppression of the Th1 immune response what results in an increased susceptibility to the infection. This is supported by Th2 modulation of the host immunity with heavy metals. Another possibility is that viable migrating larvae lead to the preferential stimulation of the Th2 response leading to the parasite evasion and increasing their survival by further down-regulation of the Th1 type of parasite specific responses (Lewis