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Effect of thimerosal on the murine immune system: especially induction of systemic autoimmunity
Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology . Linköping University, Faculty of Health Sciences.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The organic mercury compound ethylmercurithiosalicylate (thimerosal), an antiseptic and a preservative, has recently raised public health concern due to its presence in vaccines globally. Thimerosal dissociates in the body to thiosalicylate and ethyl mercury (EtHg), which is partly converted to inorganic mercuric mercury (Hg2+). The immunosuppressive, immunostimulatory, and de novo autoimmunogen effect of thimerosal in mice, as well as the accelerating/aggravating effect on spontaneous systemic autoimmunity including dose-response aspects were the subject of this thesis.

Thimerosal perorally (590 μg Hg/kg body weight (bw)/day) to genetically susceptible (H-2s) mice caused immunosuppression during the first week with reduction of the total number of splenocytes, T- and B-cells. The suppression lasted 2 weeks for CD4+ cells, but was superseded by a strong immunostimulation/proliferation including T- as well as B-cells, and polyclonal B-cell activation (PBA). Antinuclear antibodies targeting the 34-kDa nucleolar protein fibrillarin (AFA) appeared after 10 days, followed by renal mesangial and systemic vessel wall immune-complex (IC) deposits. The Lowest Observed Adverse Effect Level (LOAEL) was in the order AFA = glomerular and splenic vessel wall deposits < hyperimmunoglobulinemia < PBA. The LOAEL for AFA was 118 μg Hg/kg bw/day. The LOAEL for the different parameters of this thimerosal-induced systemic autoimmune condition (HgIA) was 3-11-fold higher compared with HgIA induced by HgCl2. The thimerosal-induced HgIA shared with HgCl2 a significant dose-response relationship, and requirement for: T-cells, the costimulatory factor CD28, the IFN-γ/IFN-γ-receptor pathway,but not IL-4. The mRNA expression in lymph nodes of IL-2, IFN-γ, IL-4, and IL-15 was significantly increased but not delayed compared with HgCl2.

Treatment with the ubiquitous organic Hg compound methyl Hg using equimolar doses of Hg (533 μg Hg/kg bw/day) caused a transient immunosuppression, followed by a weak immunostimulation and AFA. The IgG AFA isotypes induced by the organic Hg compounds MeHg and EtHg were stable and dominated by a Th1-like pattern over a broad time- and dose range. Treatment with inorganic HgCl2 caused a dose- and time-dependent pattern of IgG AFA isotypes. Low doses favored a Th1-like pattern, a high dose a balanced or Th2-like pattern. Middle-range doses showed initially a Th1-like pattern which gradually evolved into a balanced or Th2-like pattern. The qualitative difference in IgG AFA isotypes between organic and inorganic Hg may be due to differences in activation and/or suppression of T-helper cell subsets or factors influencing the Th1/Th2-function. Speciation of the renal Hg2+ concentration and comparison with the threshold dose for induction of AFA by HgCl2 showed that even with the lowest doses of thimerosal and MeHg used in this thesis, the AFA response might from a dose threshold point of view have been caused by conversion of the organic Hg species to Hg2+.

Primary treatment with inorganic Hg (HgCl2) accelerates/aggravates murine systemic autoimmunity, both spontaneous (genetic) and induced by other means. This capacity was assessed for thimerosal over a broad dose range using the (NZB X NZW)F1 hybrid mouse model. Significantly increased antinuclear antibodies (ANA) was seen after 4-7 weeks treatment (LOAEL 147 μg Hg/kg bw/day), and the response was dose-dependent up to 13 weeks. Renal mesangial and systemic vessel walls deposits similar to those in de novo HgIA were present after 7 weeks treatment. Twenty-two to 25 weeks treatment with thimerosal caused, in a dose-dependent fashion (LOAEL 295 μg Hg/kg bw/day), relocalization of the spontaneously developing glomerular IC deposits from the capillary vessel walls to the mesangium, which attenuated histological kidney damage and proteinuria, and increased survival. Thimerosal caused systemic vessel wall IC-deposits over a broad dose range: the Low Observed Adverse Effect Level (LOAEL) for renal and splenic vessel wall IC deposits was 18 and 9 μg Hg/kg bw/day, respectively. The No Observed Adverse Effect Level (NOAEL) could not be determined for the latter, since deposits were present even with the lowest dose used.

Thimerosal causes in genetically susceptible mice an initial, transient immunosuppression which is superseded by a strong immunostimulation and systemic autoimmunity, sharing many characteristics with the HgIA induced by inorganic HgCl2. The IgG AFA isotype pattern is however qualitatively different, and the threshold dose substantially higher. In contrast, long-term treatment with thimerosal induces systemic vessel wall IC-deposits also using doses below those needed to induce HgIA de novo in H-2s mice.

Place, publisher, year, edition, pages
Institutionen för molekylär och klinisk medicin , 2006.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 935
Keyword [en]
thimerosal, ethylmercury, autoimmunity, mice, immunosuppression, Th1, Th2
National Category
Pathobiology
Identifiers
URN: urn:nbn:se:liu:diva-6315ISBN: 91-85497-71-1 (print)OAI: oai:DiVA.org:liu-6315DiVA: diva2:21752
Public defence
2006-02-17, Linden, Entré 65, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2006-04-21 Created: 2006-04-21 Last updated: 2009-08-22
List of papers
1. Dose-response study of thimerosal-induced murine systemic autoimmunity
Open this publication in new window or tab >>Dose-response study of thimerosal-induced murine systemic autoimmunity
2004 (English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, Vol. 194, no 2, 169-179 p.Article in journal (Refereed) Published
Abstract [en]

The organic compound ethylmercurithiosalicylate (thimerosal), which is primarily present in the tissues as ethylmercury, has caused illness and several deaths due to erroneous handling when used as a disinfectant or as a preservative in medical preparations. Lately, possible health effects of thimerosal in childhood vaccines have been much discussed. Thimerosal is a well-known sensitizing agent, although usually of no clinical relevance. In rare cases, thimerosal has caused systemic immune reactions including acrodynia. We have studied if thimerosal might induce the systemic autoimmune condition observed in genetically susceptible mice after exposure to inorganic mercury.

A.SW mice were exposed to 1.25–40 mg thimerosal/l drinking water for 70 days. Antinucleolar antibodies, targeting the 34-kDa protein fibrillarin, developed in a dose-related pattern and first appeared after 10 days in the two highest dose groups. The lowest observed adverse effect level (LOAEL) for antifibrillarin antibodies was 2.5 mg thimerosal/l, corresponding to an absorbed dose of 147 μg Hg/kg bw and a concentration of 21 and 1.9 μg Hg/g in the kidney and lymph nodes, respectively. The same LOAEL was found for tissue immune-complex deposits. The total serum concentration of IgE, IgG1, and IgG2a showed a significant dose-related increase in thimerosal-treated mice, with a LOAEL of 5 mg thimerosal/l for IgG1 and IgE, and 20 mg thimerosal/l for IgG2a. The polyclonal B-cell activation showed a significant dose–response relationship with a LOAEL of 10 mg thimerosal/l. Therefore, thimerosal induces in genetically susceptible mice a systemic autoimmune syndrome very similar to that seen after treatment with inorganic mercury, although a higher absorbed dose of Hg is needed using thimerosal. The autoimmune syndrome induced by thimerosal is different from the weaker and more restricted autoimmune reaction observed after treatment with an equipotent dose of methylmercury.

Keyword
Thimerosal, Ethylmercury, Mice, Autoimmunity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13815 (URN)10.1016/j.taap.2003.09.006 (DOI)
Available from: 2006-04-21 Created: 2006-04-21 Last updated: 2009-08-18
2. Immunosuppressive and autoimmune effects of thimerosal in mice
Open this publication in new window or tab >>Immunosuppressive and autoimmune effects of thimerosal in mice
Show others...
2005 (English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, Vol. 204, no 2, 109-121 p.Article in journal (Refereed) Published
Abstract [en]

The possible health effects of the organic mercury compound thimerosal (ethylmercurithiosalicylate), which is rapidly metabolized to ethylmercury (EtHg), have recently been much debated and the effect of this compound on the immune system is largely unknown. We therefore studied the effect of thimerosal by treating A.SW (H-2s) mice, susceptible to induction of autoimmunity by heavy metals, with 10 mg thimerosal/L drinking water (internal dose ca 590 μg Hg/kg body weight/day) for up to 30 days. The lymph node expression of IL-2 and IL-15 mRNA was increased after 2 days, and of IL-4 and IFN-γ mRNA after 6 and 14 days. During the first 14 days treatment, the number of splenocytes, including T and B cells as well as Ig-secreting cells decreased. A strong immunostimulation superseded after 30 days treatment with increase in splenic weight, number of splenocytes including T and B cells and Ig-secreting cells, and Th2- as well as Th-1-dependent serum immunoglobulins. Antinucleolar antibodies (ANoA) targeting the 34-kDa nucleolar protein fibrillarin, and systemic immune-complex deposits developed. The H-2s strains SJL and B10.S also responded to thimerosal treatment with ANoA. The A.TL and B10.TL strain, sharing background genes with the A.SW and B10.S strain, respectively, but with a different H-2 haplotype (t1), did not develop ANoA, linking the susceptibility to H-2. Thimerosal-treated H-2s mice homozygous for the nu mutation (SJL-nu/nu), or lacking the T-cell co-stimulatory molecule CD28 (B10.S–CD28−/−), did not develop ANoA, which showed that the autoimmune response is T-cell dependent. Using H-2s strains with targeted mutations, we found that IFN-γ and IL-6, but not IL-4, is important for induction of ANoA by thimerosal. The maximum added renal concentration of thimerosal (EtHg) and inorganic mercury occurred after 14 days treatment and was 81 μg Hg/g. EtHg made up 59% and inorganic mercury 41% of the renal mercury. In conclusion, the organic mercury compound thimerosal (EtHg) has initial immunosuppressive effects similar to those of MeHg. However, in contrast to MeHg, thimerosal treatment leads in genetically susceptible mice to a second phase with strong immunostimulation and autoimmunity, which is T-cell dependent, H-2 linked and may at least partly be due to the inorganic mercury derived from the metabolism of ethyl mercury.

Keyword
Thimerosal; Ethylmercury; Mice; Immunosuppression; Autoimmunity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13816 (URN)10.1016/j.taap.2004.08.019 (DOI)
Available from: 2006-04-21 Created: 2006-04-21
3. The autoimmunogen effect of the organic mercury species methyl mercury and ethyl mercury
Open this publication in new window or tab >>The autoimmunogen effect of the organic mercury species methyl mercury and ethyl mercury
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-13817 (URN)
Available from: 2006-04-21 Created: 2006-04-21 Last updated: 2010-01-13
4. Alteration of the spontaneous systemic autoimmune disease in (NZB x NZW)F1 mice by treatment with thimerosal (ethyl mercury)
Open this publication in new window or tab >>Alteration of the spontaneous systemic autoimmune disease in (NZB x NZW)F1 mice by treatment with thimerosal (ethyl mercury)
2006 (English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, Vol. 214, no 1, 43-54 p.Article in journal (Refereed) Published
Abstract [en]

Inorganic mercury may aggravate murine systemic autoimmune diseases which are either spontaneous (genetically determined) or induced by non-genetic mechanisms. Organic mercury species, the dominating form of mercury exposure in the human population, have not been examined in this respect. Therefore, ethyl mercury in the form of thimerosal, a preservative recently debated as a possible health hazard when present in vaccines, was administered in a dose of 0.156–5 mg/L drinking water to female (NZB × NZW)F1 (ZBWF1) mice. These mice develop an age-dependent spontaneous systemic autoimmune disease with high mortality primarily due to immune-complex (IC) glomerulonephritis. Five mg thimerosal/L drinking water (295 μg Hg/kg body weight (bw)/day) for 7 weeks induced glomerular, mesangial and systemic vessel wall IC deposits and antinuclear antibodies (ANA) which were not present in the untreated controls. After 22–25 weeks, the higher doses of thimerosal had shifted the localization of the spontaneously developing renal glomerular IC deposits from the capillary wall position seen in controls to the mesangium. The altered localization was associated with less severe histological kidney damage, less proteinuria, and reduced mortality. The effect was dose-dependent, lower doses having no effect compared with the untreated controls. A different effect of thimerosal treatment was induction of renal and splenic vessel walls IC deposits. Renal vessel wall deposits occurred at a dose of 0.313–5 mg thimerosal/L (18–295 μg Hg/kg bw/day), while splenic vessel wall deposits developed also in mice given the lowest dose of thimerosal, 0.156 mg/L (9 μg Hg/kg bw/day). The latter dose is 3- and 15-fold lower than the dose of Hg required to induce vessel wall IC deposits in genetically susceptible H-2s mice by HgCl2 and thimerosal, respectively. Further studies on the exact conditions needed for induction of systemic IC deposits by low-dose organic mercurials in autoimmune-prone individuals, as well as the potential effect of these deposits on the vessel walls, are warranted.

Keyword
Thimerosal; Mice; Autoimmunity; Immune-complex; (NZB × NZW)F1 mice
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13818 (URN)10.1016/j.taap.2005.12.004 (DOI)
Available from: 2006-04-21 Created: 2006-04-21

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