Superfund Research Program
Biomarkers of Chromogenic Solvent Exposure & Neurodegeneration
Recognition that certain straight-chain (aliphatic) organic solvents have the potential to cause peripheral neuropathy is longstanding. Equally well established was that aromatic (ring-structure) solvents lack this property. Recent research at Oregon Health & Science University (OHSU) has shown this dogma to be incorrect. The OHSU team has shown that certain aromatic hydrocarbons are not only potent neuropathic agents but also have the remarkable property of turning tissue blue and urine green!
That aromatic solvents could be both chromogenic and neurotoxic first surfaced in the early 1900s when children and adults were reported to excrete green urine and develop acute central nervous toxicity after either domestic and occupational exposure to tetralin-containing waxes or varnishes. By 1960, several widely-used aromatic solvents were shown to be chromogenic but their neurotoxic potential was not assessed. Later, around 1980, a tetralin derivative used in fragrances and foods was withdrawn after it was shown to cause spinal cord and peripheral nerve damage in rats. These and a number of other tetralin compounds can now be found in trace quantities in water, sludge and fish.
Many widely used non-chlorinated aromatic solvents (benzene, o-xylene, diethylbenzene, triethylbenzene) are reported to have chromogenic properties, but these have not been assessed with modern analytical methods. Solvents such as these are among the most common contaminants of soil and water at Superfund sites. If chromogenicity and neurotoxicity are linked, chromogenic solvents should be tested for neurotoxic potential.
Knowledge about the mechanism of the chromogenic effects could provide valuable insights into the fundamental mechanisms of the neurotoxicity. Further, knowledge that the chromophore appears in tissues and is excreted in urine before neurological damage occurs could be applied in the development of biomarkers of exposure to the chromogenic neurotoxic compounds. A clear understanding of the relationship could be valuable to risk assessors and public health officials.
OHSU scientists Dr. Mohammad Sabri and Dr. Peter Spencer lead a research team working to delineate the relationship between the chromogenicity and neurotoxicity of non-chlorinated organic hydrocarbon solvents. The work is funded by the NIEHS Superfund Basic Research Program (SBRP) in conjunction with the NIEHS NeuroToxicogenomics Research Center, both of which are housed in the University's Center for Research on Occupational and Environmental Toxicology. The studies focus on the chromogenic benzene derivative 1,2-diethylbenzene (1,2-DEB) and its metabolite 1,2-diacetylbenzene (1,2-DAB). OHSU researchers have shown that the colorless 1,2-DAB forms a blue pigment on contact with proteins, skin and other tissues. Repeated exposure to 1,2-DAB produces limb weakness in rats related to the development of intraspinal and spinal root axonal swellings containing excessive numbers of neurofilaments (NF). A similar pattern of intraspinal pathology is reported in amyotrophic lateral sclerosis (Lou Gehrig's disease); however, while motor neurons degenerate in Lou Gehrig's disease, this has not been observed in 1,2-DAB-treated rats.
Further work at OHSU showed that 1,2-DAB reacts preferentially with protein constituents of neurofilaments to form protein adducts of high molecular weight (i.e. polymers). Similar changes occur with another neurotoxic solvent metabolite, 2,5-hexanedione (2,5-HD), the agent responsible for peripheral neuropathy in humans occupationally exposed to the aliphatic organic solvents n-hexane or methyl n-butyl ketone. Structurally, both 1,2-DAB and 2,5-HD are gamma-diketones. Based on their ability to form protein adducts, 2,5-HD has a lower neurotoxic potency than 1,2-DAB.
The OHSU scientists are investigating the structure-activity relationships for the chromogenic and neurotoxic properties of the aromatic and aliphatic solvent metabolites. They have compared the reactivity of diacetylbenzene (DAB) and hexanedione (HD) isomers with amino acids and proteins (including neurofilament proteins) in vitro and in vivo. They found that:
- 1,2-DAB forms blue-purple pigments with amino acids and proteins, polymerizes neurofilament proteins, and induces tissue discoloration, behavioral changes and peripheral neuropathy in rats.
- 1,3-DAB (which is not a gamma-diketone) is non-reactive with proteins, non-chomogenic and non-neurotoxic.
- 2,5-HD, but not 2,4-HD, is weakly chromogenic, reacts with proteins and cross links neurofilaments at higher concentrations than 1,2-DAB; and induces peripheral neuropathy in rats with neurofilament swellings in nerves rather than in spinal cord or spinal roots.
As part of their SBRP-funded project, the OHSU team is collaborating with researchers at Battelle Research Institute, which directs the Pacific Northwest National Laboratories, to understand the relationship between the protein reactivity and neurotoxic properties of aromatic and aliphatic solvent metabolites. Additional research on the neurofilament targets of gamma-diketones is underway with collaborating scientists at Oregon State University, which houses an NIEHS-funded Environmental Toxicology Center.
The research conducted by the OHSU researchers has led to significant new findings with respect to the role of molecular structure in the genesis of peripheral neuropathy and the relationship between chromogenicity and neurotoxicity. Specifically, the OHSU team has shown that:
- 1,2-DAB is a potent neurotoxic aromatic hydrocarbon. Isomers (1,3-DAB and 1,4-DAB) that lack the gamma-diketone structure lack this property.
- While aromatic and aliphatic gamma-diketones share neurotoxic mechanisms, the aromatic compound has a much higher neurotoxic potency in rodent studies.
- Because chromogenicity and neurotoxicity are closely related properties, other reportedly chromogenic solvents in widespread use should be tested for neurotoxic properties.
- Chromogenicity (green urine) precedes neurotoxicity and might therefore be a useful biomarker for exposure to organic solvents with neurotoxic potential.
For More Information Contact:
Mohammad I. Sabri
Oregon Health and Science University
Center for Research on Occupational and Environmental Toxicology
3181 S.W. Sam Jackson Park Road, L606
Portland, Oregon 97239-3098
To learn more about this research, please refer to the following sources:
- Kim M, Spencer PS, Sabri MI. 2002. Amino acid and protein targets of 1,2-diacetylbenzene, a potent aromatic gamma-diketone that induces proximal neurofilamentous axonopathy. Toxicol Appl Pharmacol 183(1):55-65. PMID:12217642
- Zhan C, Dixon DA, Spencer PS, Kim M, Sabri MI. 2002. Theoretical determination of chromophores in the chromogenic effects of aromatic neurotoxicants. J Am Chem Soc 124(11):2744-2752. PMID:11890826
- Kim M, Sabri MI, Miller VH, Kayton RJ, Dixon DA, Spencer PS. 2001. 1,2-diacetylbenzene, the neurotoxic metabolite of a chromogenic aromatic solvent, induces proximal axonopathy. Toxicol Appl Pharmacol 177(2):121-131. PMID:11740911
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