1. Neurotoxic Effects of Solvents
William Boyes
Neurotoxicology Division
National Health and Environmental Effects Research Laboratory
Office of Research and Development, EPA

2. Chemical Structures Benzene Toluene
Tetrachloroethylene (Perchloroethylene) or “Perc”
Trichloroethylene (TCE)

3. Solvents Lipophilic Volatility Organic chemical composition
Distribute to body lipid-rich tissues
Easily cross lipid membranes & barriers
Volatility
Inhalation is a significant route of exposure
Delivery to tissues depends upon the blood:tissue partition coefficient

4. Solvent Neurotoxicity
Acute
Chronic
CNS depression
Euphoria
Sensory, cognitive, motor deficits
Reversible after exposure
Causes accidents or injuries
Good animal models
Several mechanistic targets
Organic brain disorder
Originally observed in Scandinavian painters
Sensory, cognitive, motor deficits
Years of high level exposure
No good animal models
Mechanisms unknown

5. Volatile Organic Compounds
Uses include:
Organic solvents
Cleaning & degreasing
Gasoline
Paints & glues
Dry cleaning
Printing
Paint strippers, nail polish remover
Microelectronic manufacture
Widely used in industry and commerce
Emission Sources
Factories & chemical plants
Smaller shops
Mobile sources
Indoor air sources
Consumer products
Pesticide “inerts”
Drinking water
Hazardous waste sites

6. The Washington Post, Aug. 14, 2002
Some Current Concerns
Upsets
Fuels
Dry Cleaners
The Washington Post, Aug. 14, 2002
Public Citizen, 2005

7. Mechanism of Action Two hypotheses: Membrane fluidity
Membrane proteins
Antikowiak, Naturwissenschaften, 88: , 2001

8. Oocyte Removal Surgery
Female
Xenopus
Laevis
Frog
cDNA
Cut with Restriction Enzymes
In Vitro Transcription
mRNA
Oocyte Removal Surgery
Linearized
DNA
Separated
Stage VI, V
Oocytes
Incubate 2-7 days at 18oC
Oocyte Injections
Voltage
Current
EUREKA
Currents!!!
ACh
2 sec
50 nA
Clamp at appropriate
test potential
Two-Electrode Voltage Clamp

9. Toluene Reversibly Inhibits nAChRs
5 sec
ACh
1 mM TOL
a7 - human
100 nA
2 sec
a7-rat
20 sec
200 nA
a4b2-human
50 nA
a4b2-rat
Bale et al., 2005

10. nAChR Inhibition by Toluene: Concentration-Dependent and Species-Independent
100
100
a7-
human 75
a7-
rat 75
% Inhibition 50
% Inhibition 50
a4b2-human 25 25
a4b2-rat 10 -4 10 -3 10 -2 10 -4 10 -3 10 -2
[Toluene] M
[Toluene] M
Modified from Bale et al., 2005

11. Solvents & Ion Channel Function
Bushnell et al., 2005

12. Chronic Solvent Encephalopathy?
Scandinavian painters and other workers
Chronic exposure
a variety of impairments of mood and intellectual function leading eventually to dementia
Early studies confounded
Poorly matched controls
Poor documentation of exposure history
More recent studies show
Increased reaction times
Poor visual function
Impaired auditory thresholds
Impaired motor skills
Impaired performance of cognitive and memory tasks

13. Chronic Toxic Encephalopathy in a Painter Exposed to Mixed Solvents
(Feldman et al., Environmental Health Perspectives 107) 1999
However:
Limitations in design and strength of association make it impossible to draw reliable conclusions regarding nervous system damage from organic solvents. (Ridgeway et al., 2003)

14. Neurobehavioral Deficits in Solvent-Exposed Painters
Grosch et al., Am J Indust Med, 30: (1996)
Long-term chronic or acute high dose exposure can produce cognitive and behavioral changes (White and Proctor, 1997)

15. Weight of Evidence Toluene Human Neurotoxicity

16. Toluene Present in paints, glues, gasoline and many other products
Subject of over 40 EPA Maximum Achievable Control Technology (MACT) / residual risk assessments
The substance of choice for glue sniffers
Toluene
Benzoic acid
Hippuric acid
CH3
COOH
COOGly
glycine

17. Toluene and Momentary Brain Concentration
Bushnell et al., 2007

18. Toluene Abuse Neuropathic effects in humans
Following repeated solvent abuse
Very high dose levels
Cerebellar damage
Cerebral atrophy
Multiple symptoms of dementia
Confounded by hypoxia and other exposures

19. Toluene at High Doses Causes Outer Haircell Damage in Chochlea
Johnson (1993)

20. Ethanol (Alcohol) CNS depressant
CH3—CH2—OH
CNS depressant
Legal definition of inebriation based on BACs
Often 0.1% (100 mg/100 ml) (0.08 % in NC)
Acute exposure
Euphoria
Loss of inhibitions / poor judgment
Loss of balance & motor coordination
Impaired vision & visual/motor function
Ataxia, nausea, vomiting
Unconsciousness

21. Ethanol Metabolism CH3—CH2—OH CH3—CHO + NADH NAD Ethanol Acetaldehyde
Alcohol dehydrogenase
CH3—CH2—OH
CH3—CHO
+ NADH
NAD
Ethanol
Acetaldehyde
< 10%
Catalase
H2O2
10-30% of metabolized ethanol
NADPH
Rate of metabolism is ~ mg%/hr
< 20%
Microsomal ethanol oxidizing system (MEOS) = CYP2E1

22. Alcohol dehydrogenase (ADH)
Ethanol Metabolism
Alcohol dehydrogenase (ADH)
CH3—CH2—OH
CH3—CHO
+ NADH
NAD
Ethanol
Acetaldehyde
NAD
Acetaldehyde dehydrogenase
(ALDH)
ADH
males > females
ALDH isozymes
inactive variant in %50 Asians
Variant form in Native Americans
inhibited by Disulfiram
CH3—COO-
+ NADH
Acetic acid

23. Chronic Ethanol Toxicity
Alcoholism
High % of calories from alcohol
Thiamine deficient
Wernicke's encephalopathy
Damage to multiple brain areas
Impaired cognition, motor function
Korsakoff's psychosis
Acute & Chronic
100,000 premature deaths / year in U.S.

24. Fetal Alcohol Syndrome (FAS)
Characterized by:
Mental retardation
Microcephaly
Irritability
Reduced birth weight
Poor muscle coordination
Cranio/Facial anomalies
Mechanism poorly understood
FAS: 0.2 to 1.5 per 1,000 live births in US (CDC)
Other FASDs approximately three times as often as FAS
Drinking and heavier drinking remain at high levels among women of child-bearing age (Caetano et al., 2006)

25. Methanol (Wood Alcohol)
CH3—OH
Clinical signs (people)
Multiphasic syndrome
Early – like ethanol (Central nervous system depression, weakness, headache, vomiting)
Mid- asymptomatic period (12-24 hr)
Late - Severe metabolic acidosis, optic disc edema, and bilateral necrosis of the putamen
Other adverse effects of methanol in humans include minor skin and eye irritation
Formic acid is the toxic metabolite of methanol.
Accounts for the metabolic acidosis and blindness seen in people following methanol poisoning

26. Methanol Metabolism (Primates)
Alcohol dehydrogenase
CH3—OH
CH2O
+ NADH
NAD
Methanol
Formaldehyde
NAD
Formaldehyde dehydrogenase
Tetrahydrofolate
CO2
HCOO-
+ NADH
Low
Formic acid

27. Methanol Metabolism (Rodents)
Catalase
CH3—OH
CH2O
Methanol
Formaldehyde
NAD
Formaldehyde dehydrogenase
Tetrahydrofolate
CO2
HCOO-
+ NADH
HIGH
Formic acid
Folate deficiency increases the sensitivity of methanol in rodents. Intraretinal metabolism may be important.

28. Methanol Visual Toxicity
Red light reverses methanol toxicity via action on mitochondrial cytochrome ?? Energy transfer
Eells et al., PNAS 2003

29. n-Hexane and Methyl n-butyl ketone
Neurotoxicity
Sensorimotor polyneuropathy
Sensory numbness and paresthesia
Distal nerves affected first
Clinical signs often delayed for months
Axonal swelling and secondary demyelination
2,5-hexanedione is common toxic metabolite

30. Spencer & Schaumburg, 2000

31. Peripheral Neuropathy

32. Sources & Readings
Anthony, DC., Montine, T.J., Valentine W.M., and Graham, D.G. Toxic responses of the nervous system. In Casarett and Doull’s Toxicology: the Basic Science of Poisons, Sixth Edition. McGraw-Hill Medical Publishing Division, New York, pp , 2001
Bushnell, P.J., Shafer, T.J., Bale, A.S., Boyes, W.K., Simmons, J.E., Eklund, C. and Jackson, T.L. Developing an exposure-dose-response model for organic solvents: overview and progress on in vitro models and dosimetry. Environmental Toxicology and Pharmacology, 19: 607–614, 2005.
Benignus, V.A., Bushnell, P.J. and Boyes, W. K. Toward cost-benefit analysis of acute behavioral effects of toluene in humans. Risk Analysis, 25 (2), , 2005.
Bruckner, J.V. and Warren, D.A., W.K. Toxic effects of solvents and vapors. In Casarett and Doull’s Toxicology: the Basic Science of Poisons, Sixth Edition. McGraw-Hill Medical Publishing Division, New York, pp , 2001
Schreiber, J S; Hudnell, H K; Geller, A M; House, D E; Aldous, K M; Force, M E; Langguth, K W; Prohonic, E J; Parker, J C (2002) Apartment residents’ and day care workers’ exposures to tetrachloroethylene and deficits in visual contrast sensitivity. Environ Health Perspect 110:655–664.
Arlien-Søborg, P. (1992). Solvent Neurotoxicity. CRC Press, Boca Raton FL.
Spencer PS., and Schaumburg H.H. Experimental and Clinical Neurotoxicology, 2nd Edition, Oxford University Press, 2000.