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The blood-brain barrier in toxicology

Ursula Gundert-Remy1* and Ralf Stahlmann2
  • 1 Federal Institute for Risk Assessment, Department of Toxicology, Germany
  • 2 Department Toxicology,Charité, Garystrasse 5, Germany

There is increasing awareness of the potential importance of the blood-brain barrier (BBB) in toxicology and in risk assessment of environmental stressors. This contribution deals with some aspects of the BBB in toxicology with the following topics being discussed (i) specific considerations on transport, (ii) test methods in toxicology related to the exposure of the brain, (iii) substances of concern including (iiia) metals and metal-induced CNS diseases (iiib) cholinesterase inhibitors (pesticides, warfare agents) (iiic) paraquat (herbicide) (iiid) toxins.

Specific considerations on transport
The processes governing the entrance and release of xenobiotics to and from brain cells through the BBB, are physiologically defined biological processes which apply in toxicology as in pharmacology. Hence, in this aspect there is no differentiation between drugs and toxicants. However, the specific metal transporters play a role concerning the uptake of metals by brain cells, which in toxicology may be important as some of the metals have been imputed as a cause for neurotoxicity and CNS disease. The metal transporters are known to mediate the influx of aluminium, copper, zinc and other metals (Pelkonen et al., 2008).

Relevant toxicological test methods to evaluate whether a substance crosses the BBB
The regulatory tests, which have to be performed in order to ensure that an industrial chemical, a pesticide, herbicidal or biocidal substance can be safely applied, are defined by a consensus at the international level of the organisation for economic cooperation and development (OECD). In OECD tests 407and 408, which apply to all substances as far as repeated dose toxicity testing concerns, sensory reactivity to stimuli of different types and motor activity assessment provide evidence for an effect of the substance on the CNS, which is an indirect indication that the substance crosses the BBB. For pesticides of the organophosphate type, OECD test 418 (acute exposure) and OECD test 419 (repeated dosing) are performed to detect behavioural abnormalities and impairment in forced motor activity. For substances of concern, the OECD test 424 (Specific Neurotoxicity Study) is specifically suited to detect CNS impairment monitoring activity level, coordination of movement, and presence of clonic or tonic movements. In order to detect developmental neurotoxicity the OECD test focuses on the study of gross neurologic and behavioural abnormalities, on behavioural ontogeny, motor activity and sensory function. In addition, learning and memory tests are to be performed.
Thus, whereas specific toxicokinetic studies to evaluate whether a substance is able to cross the BBB are not to be performed, toxicodynamic studies are requested for specific types of substances and specific exposures with the aim to detect toxic effects on the brain. Thus, there is indirect proof that a substance can cross the BBB if a test turns out to indicate brain toxicity by a given substance.

Substances of concern
Metals and metal-induced diseases
The importance of metals results from environmental exposure and from observational studies on associations between exposure and diseases of the CNS such as Alzheimer and Parkinson.

Mercury
The long occupational history has identified the major signs and symptoms of severe poisoning from inhaled mercury. Tremor and psychological disturbances are the main features. Mercury-induced tremor in milder cases is intentional, which occurs during guided movements (finger-to-nose test), but in more severe cases tremor becomes postural (tremor in the extended arm). Numerical values are obtained for tremor, skill, coordination, and nerve conduction velocity. Workers who were examined some 25 to 30 years after exposure and had experienced peak urine levels of about 500 μg Hg/L still demonstrated adverse effects in the nervous system. These included decreased coordination, increased tremor, and decreased sensation when compared with controls. The dental profession has been the subject of many studies on effects of mercury (Clarkson and Magos, 2006). In addition, mercury has been imputed to be related to the development of Alzheimer's disease (AD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) (Mutter et al. 2007). The mechanism by which mercury is entering the brain and evoking its toxic effect has been elucidated. Elemental mercury is taken up as vapour and crosses readily the alveolar membranes in the lung to enter systemic circulation. It diffuses across the BBB in the brain tissue where it is oxidized to the divalent Hg++ cation by the catalase II-hydrogen peroxide system and reacts with –SH containing ligands being fixed to the tissue (Aschner and Aschner, 1990).

Lead
Primarily due to its ability to substitute for calcium ions (Ca2+), Pb2+ crosses rapidly the BBB and concentrates in the brain. Picomolar concentrations of Pb2+ can replace micromolar concentrations of Ca2+ in a protein kinase enzyme assay, a calcium dependent process. Thus, at the functional level of the BBB, the ability of lead to mimic or to mobilize calcium and protein kinase C could alter the behaviour of endothelial cells in the immature brain and disrupt the BBB. Lead has effects on synapse formation, neuronal growth, and organisation of ion channels, neurotransmitter production and neurotransmission. In children, a whole array of symptoms have been noted including learning disabilities, reduced fine motor skills, and emotional/social behaviour deficits. Even attention deficit hyperactivity disorder (ADHS) has been attributed to brain overload with lead. In adults, lead causes peripheral neuropathy and effects on the CNS with loss of memory function, and shrinking of brain volume as demonstrated by magnetic resonance imaging (reviewed by Sanders et al., 2009).

Silver
Exposure to silver is increasing because of silver Nan particles in consumer products. A recent in vitro study gives rise to concern as it is known that Nan particles can cross the BBB (Oberdörster et al., 2004). In this in vitro study in undifferentiated PC12 cells, Ag+ (10 μM) inhibited DNA and protein synthesis. With the onset of cell differentiation Ag+ -treatment showed impaired neurite formation, redirecting the differentiation into a dopamine phenotype and suppressing development into the acetylcholine phenotype. In differentiating cells, low Ag+ exposure (1 μM) increased cell numbers by suppressing ongoing cell death and impairing differentiation into both neurotransmitter phenotypes. The conclusion from this study is that silver has the potential to evoke developmental neurotoxicity, thus rising concern with respect to the use of silver nanoparticles (Powers et al., 2010).

Pesticides
Pesticides are a toxicologically heterogeneous group of substances. Among them, organophosphates have been always in the focus of attention, particularly because in the past organophosphate substances were used as warfare agents.

Cholinesterase inhibitors/Organophosphates
The acetyl cholinesterase blocking substance phosalone has been demonstrated to impair the function of the BBB in rats by increasing the permeability for sucrose, indicating a role of acetyl cholinesterase in maintaining the barrier function (Bharavi and Reddy, 2005).
Effects of chronic chlorpyrifos exposure on cognitive function in adult and developing animals have been reviewed by Eaton et al (2008). No effects on learning or memory were observed in rats that received chlorpyrifos continuously at the same dose for one year. However, animals that received additional high doses of chlorpyrifos did exhibit deficits in learning. Hence, persistent cognitive impairment may follow if chlorpyrifos exposure inhibits brain cholinesterase activity in acute doses sufficient to induce signs of neurological toxicity. Behavioural effects were clearly evident during periods of significant inhibition of brain acetyl cholinesterase. Several investigators have shown that young animals are more susceptible than adults to the acute toxicity of chlorpyrifos. Profound changes were found in muscarinic receptors, acetylcholine synthesis, and adenylate cyclase after pre-treatment with chlorpyrifos. Of particular interest is a study in which the effects of acute and repeated chlorpyrifos administration were compared in neonatal (postnatal day 7) and adult rats. Upon acute exposure, the ED50s for brain and plasma acetyl cholinesterase inhibition were 1.5–2.9 mg/kg in neonates and 3.9–4.4 mg/kg in adults, with a ratio of NOEL values between neonates and adults of 10. This finding confirms the higher susceptibility of young rats to the acute toxicity of chlorpyrifos (see Eaton et al., 2008). Using microarrays, Slotkin et al. (2007) provided evidence for an alternate mechanism of neurotoxicity of organophosphates. In their study, chlorpyrifos and diazinon both markedly suppressed the regional expression of mRNAs encoding the fibroblast growth factor 20 (fgf 20) in the forebrain and fgf2 in the brain stem. Other fgfs (fgf11, fgf14, fgf22) were less affected, while elevated levels of brain stem fibroblast growth factor receptor 4 (fgfr4) and fgfr1 were observed. These results may indicate that organophosphates elicit developmental neurotoxicity by influencing cell growth, and not because of their cholinesterase blocking activity.

Herbicides

Paraquat
Paraquat is structurally similar to N-methyl-4-phenyl pyridinium (MPP+), an experimental agent causing Parkinson's disease. Paraquat decreases striatal dopamine levels and reduces the striatal level of tyrosine hydroxylase, thus inducing Parkinson-like dopaminergic toxicity in the brain. Using a brain microdialysis technique it was shown that paraquat could cross the BBB. Further studies showed that paraquat is possibly taken up into the brain by the neutral amino acid transport system, and is then transported into striatal, possibly neuronal, cells in a Na+ -dependent manner (Shimizu et al., 2001; Prasad et al., 2009).

Bacterial toxins
Contamination of human water sources with the cyanobacterial toxin microcystin has been shown to cause hepato- and neurotoxic effects in dialysis patients. Data in primary murine whole brain cells showed the existence of several transporters in this system. Microcystin is taken up by a specific transporter system (organic anion transporting polypeptide, Oatp). In this experimental setting microcystin causes cytotoxicity, thus providing a basis to understand the neurotoxic effect in dialysis patients (Feurstein et al., 2009).

Conclusion
In contrast to drugs, which may be beneficial if crossing the BBB, for toxicants the integrity of the BBB is a safeguard to protect against neurotoxic effects. As shown, a whole array of man-made or naturally occurring substances may cross the BBB and cause adverse effects. Stressors may critically influence the development and function of the BBB in early life stages. In later life stages, exposure to environmental stressors may have an impact on the integrity of the BBB. It is important to consider the consequences of enhancing a drug’s ability to cross the BBB because of its potential adverse effects.

References

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2. Bharavi K, Reddy KS. Effect of anticholinesterase compound phosalone on blood-brain barrier (BBB) permeability Indian J Physiol Pharmacol 49 (3), 337-340, 2005.

3. Clarkson TW, Magos L The Toxicology of Mercury and Its Chemical Compounds. Critical Reviews in Toxicology, 36:609–662, 2006

4. Eaton, DL, Daroff RB, Autrup H, Bridges J, Buffler P, Costa LG, Coyle J, McKhann G, Mobley WC, Nadel L, Neubert D, Schulte-Hermann R,Spencer PS. Review of the toxicology of chlorpyrifos with an emphasis on human exposure and neurodevelopment. Critical Reviews in Toxicology, S2:1–125, 2008

5. Feurstein D, Holst K, Fischer A, Dietrich DR. Oatp-associated uptake and toxicity of microcystins in primary murine whole brain cells. Toxicology and applied pharmacology 234, 247-255, 2009.

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9. Powers CM, Wrench N, Ryde IT, Smith AM, Seidler FJ, Slotkin TA Silver impairs neurodevelopment: Studies in PC12 cells. Environmental Health Perspectives 119 (1), 2010.

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13. Slotkin TA, Seidler FJ, Fumagalli F. Exposure to organophosphates reduces the expression of neurotrophic factors in neonatal rat brain regions: similarities and differences in the effects of chlorpyrifos and diazinon on the fibroblast growth factor superfamily. Environ Health Perspect 115:909–916 (2007).

Conference: Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU, Brussels, Belgium, 11 Feb - 12 Feb, 2010.

Presentation Type: Oral Presentation

Topic: Presentations

Citation: Gundert-Remy U and Stahlmann R (2010). The blood-brain barrier in toxicology. Front. Pharmacol. Conference Abstract: Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU. doi: 10.3389/conf.fphar.2010.02.00011

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Received: 25 Feb 2010; Published Online: 25 Feb 2010.

* Correspondence: Ursula Gundert-Remy, Federal Institute for Risk Assessment, Department of Toxicology, Berlin, Germany, ursula.gundert-remy@bfr.bund.de