Research ReportNeonatal administration of a vaccine preservative, thimerosal, produces lasting impairment of nociception and apparent activation of opioid system in rats
Introduction
Thimerosal (THIM; also known as thiomerosal or sodium ethylmercurithiosalicylate), which contains about 49% of mercury (Hg) by weight, has been used as a vaccine preservative for decades without rigorous studies examining its safety in developing mammalian organism, including infants. A vast body of scientific literature provides evidence that all forms of Hg are highly toxic to animals (rev. Díez, 2009, Clarkson and Magos, 2006). THIM is biotransformed in the body to ethylmercury and subsequently also into inorganic forms of Hg (Qvarnström et al., 2003). Significant amounts of Hg have been detected in blood of human infants after inoculations with THIM-containing vaccines, with premature infants accumulating over 3 times more Hg than the mature ones (Stajich et al., 2000, Pichichero et al., 2008). Studies conducted with infant monkeys injected with THIM-containing vaccines, similar to those given to human infants, showed that after vaccination concentrations of Hg in the brain were several fold higher than those in blood and they remained markedly increased for many months (half life of Hg for in blood was about 7 days, and in the brain about 24 days) (Burbacher et al., 2005). These data suggest that Hg levels in infant brain after inoculations with THIM-containing vaccines may reach medium nanomolar concentrations, for which the neurotoxic effects have been reported in studies in vitro (Yel et al., 2005). Hence potential harm to infants from such vaccines cannot be ignored.
Many clinical and biological abnormalities (involving biochemistry, the nervous and immune systems) associated with Hg poisoning or prolonged contact resemble features of autism, hence the hypothesis was created that this neurodevelopmental disorder may result from exposure to Hg during early life (Bernard et al., 2001, Mutter et al., 2005). Several epidemiological studies linked THIM from vaccines to autism spectrum disorders, attention deficit hyperactivity disorder, learning disabilities and mental retardation (Geier and Geier, 2003, Geier and Geier, 2006, Grether et al., 2004, Desoto and Hitlan, 2007, Young et al., 2008). Other studies have not found such a relationship (Hviid et al., 2003, Madsen et al., 2003), but they were criticized for major methodological flaws or an apparent conflict of interest (Mutter et al., 2005). The fact that incidence of autism and other neurodevelopmental disorders markedly increased during the 1990s, coincidentally with the introduction of several new vaccines which contained THIM (Atladóttir et al., 2007, Merrick et al., 2004), enforces the perception that these events may be connected.
Neurodevelopmental disorders are often accompanied by self-injurious behaviors (SIB) (Schreibman, 1988, Lovell and Reiss, 1993, Symons et al., 2004). The neuropsychobiological nature of such behaviors is complex and not well understood—it might include anxiety and obsessive-compulsive elements or different modes of pain expression. Whether or not SIB are related to hyposensitivity to pain, which has been anecdotally reported in some autistic and mentally disabled individuals, is a matter of debate (Tordjman et al., 1999, Messmer et al., 2008). Nevertheless, several studies reported significant reduction of SIB after therapy with the opioid receptor antagonist, naltrexone (Barrett et al., 1989, Elchaar et al., 2006, Symons et al., 2004), albeit not all studies showed such effect (Willemsen-Swinkels et al., 1995). Apparent beneficial effect of naltrexone in treating SIB is concordant with the findings showing dysregulation of endogenous opioids in autistic and mentally retarded persons (Sahley and Panksepp, 1987, Sandman, 1988, Sandyk and Gillman, 1986, Leboyer et al., 1994, Kemp et al., 2008), suggesting that this is clinically important trait at least in some neurodevelopmental disorders.
Continuing disagreements around the issue of involvement of THIM from vaccines in neurodevelopmental disorders demand further studies, both at clinical and preclinical levels. A previously published animal study by Hornig et al. (2004) revealed a selective vulnerability of autoimmune disease-sensitive mice (SJL/J) to the neurotoxic effects of THIM doses equivalent to those used in child vaccines, but lack of such an effect in mice of other strains. However, these findings were not confirmed by work of Berman et al. (2008). This discrepancy calls for additional research.
The present preclinical study is a part of broader research project, which addresses some of the existing controversies concerning potential neurotoxic effects of THIM from vaccines in developing organisms. We are testing in animal model the hypothesis that early life exposure to this organomercurial may be responsible for persistent neurodevelopmental impairments. In this study we have focused on two major issues—the pharmacokinetics of Hg in different organs after THIM injection in suckling rats and on nociception, which is expected to reflect the status of endogenous opioids. We examined sensitivity to painful stimuli and indirectly the activity of endogenous opioids in adult rats, which as neonates were treated with THIM. In order to detect possible genetic differences in vulnerability to toxic effects of THIM we conducted the experiments with rats from two strains–Wistar and Lewis–which derive from a common original stock, but differ genetically. Lewis is characterized by a deficient hypothalamic–pituitary–adrenal (HPA) axis and thus is more prone to autoimmune diseases (Oitzl et al., 1995). Finally, we examined THIM's effect on nociception after its acute administration to adult rats.
Section snippets
THIM/Hg pharmacokinetics
The pharmacokinetic experiment measuring Hg distribution into different organs was conducted after a single THIM injection (2.9 or 5.8 mg Hg/kg, i.m.) to 10-day-old male Wistar rats. Animals were euthanized at different time points after THIM injection and organs were collected for analysis of Hg concentration. The analysis revealed that Hg from THIM accumulated in the brain, liver and kidneys (Fig. 1). In the brains of control rats there were trace amounts of Hg at the level of 0.0045 +
Discussion
This is the first study, to the best of our knowledge, which shows that administration of THIM to suckling rats causes protracted impairment in pain sensitivity. This effect was dose-dependent and present in two strains of rats, Wistar and Lewis, which showed nonetheless different sensitivity to this drug. Wistar rats reacted with analgesia to lower doses of THIM (equivalent to those used in child vaccines) than Lewis rats. There were no statistically significant differences between males and
Animals and drugs
Pregnant Wistar and Lewis rats were supplied by a breeder (Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland). The animals were kept in a room under standard environmental conditions (22 ± 1 °C, relative humidity of 60%, 12 h–12 h light–dark cycle with lights on at 07:00 h). Standard laboratory chow (Labofeed H, WPIK, Kcynia, Poland) and tap water were available ad libitum. Thimerosal was purchased from Sigma-Aldrich, Poland, and from Cfm Oscar Tropitzsch, Germany (product of
Acknowledgments
We thank Dr. Helena Gorecka and her coworkers from the Chemical Laboratory of Multi-Elemental Analyses at Wroclaw University of Technology in Poland for Hg analysis and Prof. Przemyslaw Bienkowski for constructive discussions. This publication is a part of ASTER project funded by the European Commission grant (MEXC-CT 2006-042371) and by the supplemantary funding from the Ministry of Science and Higher Education of Poland to Prof. Maria Dorota Majewska.
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