PON1 Status and Neurologic Symptom Complexes in Gulf War Veterans

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Insight/Outlook PON1 Status and Neurologic Symptom Complexes in Gulf War Veterans

Clement E. Furlong1 Departments of Genetics and Medicine, University of Washington, Seattle, Washington 98195-7360 USA

In a case of potential exposure to toxic the contention that Gulf War-related of oxidized lipids and prevention of vas- chemicals, it is often difficult to dissect neurologic syndromes were caused by cular disease (Mackness et al. 1991, the genetic, physiological, and environ- exposure to toxic chemical compounds. 1993; Watson et al. 1995). However, mental factors that contribute to illness. Do their conclusions seem to be jus- PON1 also hydrolyzes (inactivates) vari- A recently published paper (Haley et al. tified based on what is known about the ous toxic OP compounds including

1999) raises the possibility of a link be- human PON1192 protein polymor- paraoxon, chlorpyrifos oxon, diazoxon, tween detoxication enzyme genotype/ phism? Ideally, in an OP exposure sce- soman, and sarin (Davies et al. 1996). In phenotype and the risk of illness in Gulf nario, the parameters that would be use- human populations, PON1 exhibits a War veterans. This work builds on a ful for an epidemiological study are (1) substrate-dependent polymorphism; body of interesting data regarding de- an identification of the compounds to that is, different substrates (OPs) are hy- toxication enzyme polymorphisms and which the individual was exposed, (2) a drolyzed at different rates by different highlights the need to consider both ge- measure of the level of exposure, (3) a isoforms of PON1. Two genetic poly- notypic and phenotypic information in determination of the consequence of morphisms known in human popula- epidemiological studies. the exposure (e.g., toxin-mediated de- tions are the L55M substitution and the Haley et al. (1997) had previously pression of cholinesterase levels), and Q192R substitution (numbering from identified six unique symptom com- (4) the status of an individual’s genetic the initiator methionine that is the only plexes, which they suggested could rep- factors that contribute to sensitivity or amino acid removed during secretion of resent neurologic syndromes or injury resistance to the specific compound(s) this protein from the liver cells into the sustained in the Gulf War. In the present to which the individual was exposed. serum). study (Haley et al. 1999), they examined Unfortunately, for Gulf War exposures, Haley et al. (1999) noted that the relationship of polymorphisms in it is difficult to assess the level of expo- PON1R192 homozygotes or PON1Q/R192 the paraoxonase gene (PON1) to presen- sure to specific chemicals, and little, if heterozygotes were more likely to have tation of neurologic symptom com- any, information is available on cholin- neurologic symptom complexes than plexes in Gulf War veterans. Paraoxo- esterase levels before and after exposure. were individuals homozygous for nase (PON1) is a high density lipopro- Without the availability of exposure PON1Q192. In addition, they measured tein (HDL)-associated enzyme that information and cholinesterase inhibi- the serum activity of the enzymes and metabolizes oxidized lipids and also hy- tion data, the authors have focused on noted that low activity of the plasma drolyzes arylesters and a number of measurable parameters: the status of two PON1Q192 isoform correlated with ill- toxic organophosphorus (OP) com- detoxifying enzyme systems in indi- ness better than the PON1 genotype or pounds, including insecticides and viduals with symptom complexes. One the activity levels of the PON1R192 iso- nerve agents (Aldridge 1953b; Geldma- of these enzymes, butyrylcholinesterase form, total arylesterase, total paraoxo- cher-von Mallinckrodt and Diepgen (BChE), acts as a suicide trap for specific nase, or BCHE. Altogether, these results

1988; Davies et al. 1996). Haley et al. toxic OP compounds; that is, once BChE suggest that the PON1Q192 allele had a (1999) found that veterans homozygous reacts with an OP molecule, the OP is protective effect against neurologic for a specific PON1 allele (PON1Q192) not available for inhibiting cholinester- symptom complexes in Gulf War veter- were less likely to have neurologic symp- ase, but the enzyme is inactivated in the ans. Here, we will examine the available tom complexes than those possessing process (Aldridge 1953a). The second evidence as to whether it is reasonable the alternate allele (PON1R192). They enzyme, PON1, is capable of catalyti- that individuals with a specific PON1 also noted that low activity of the cally hydrolyzing a number of toxic status could be more sensitive to the ef- plasma PON1Q192 isoform distinguished organophosphates (Aldridge 1953b; fects of OP compounds than individuals ill veterans from controls even better Geldmacher-von Mallinckrodt and with a different PON1 status, and why than the PON1 genotype. According to Diepgen 1988; Davies et al. 1996). PON1 this might be so. Haley et al. (1999), these results support is tightly associated with HDL (“good Historically, it was known that one cholesterol”) particles (Blatter et al. enzyme isoform hydrolyzed paraoxon at

1 E-MAIL [email protected]; FAX: (206) 1993), and its main physiological func- a high rate and the other at a lower rate 543-0754. tion appears to be the metabolism (Geldmacher-von Mallinckrodt and

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Furlong

Diepgen 1988). Several years of research and Furlong 1999). In addition to pro- cation are shown in Figure 1. Early evi- in two laboratories, our own and Dr. viding an accurate inference of the dence showed a good correlation be-

Bert La Du’s (University of Michigan), PON1192 genotype, the two-dimensional tween PON1 levels in different species showed that the amino acid arginine at enzyme analysis also provides a measure and their resistance to specific OP com- position 192 resulted in a higher rate of of how much PON1 activity is present in pounds (Brealey et al. 1980; Costa et al. hydrolysis for paraoxon, whereas gluta- an individual’s serum (Davies et al. 1987; Furlong et al. 1989). More direct mine at this position resulted in a slower 1996; Richter and Furlong 1999). These tests of this hypothesis involved inject- rate of paraoxon hydrolysis. (Adkins et assays are similar to those developed by ing purified rabbit PON1 into rodents al. 1993; Humbert et al. 1993). The op- La Du and coworkers (Eckerson et al. and demonstrating increased resistance posite is true for soman and sarin (Dav- 1983) using phenylacetate and para- to specific OP compounds (Main 1956; ies et al., 1996). Subsequent research has oxon as the substrate pair. Costa et al. 1990; Li et al. 1993, 1995). shown that there is an association of Because PON1 levels vary among in- These experiments clearly demonstrated

PON1M55 isoform with lower levels of dividuals by at least 13-fold (Furlong et that high levels of PON1 protected PON1 activity in serum (Blatter Garin et al. 1989; Davies et al. 1996), it is as im- against cholinesterase inhibition by ex- al. 1997; Mackness et al. 1997), but this portant to consider PON1 levels as posure to the highly toxic oxon forms of is not an absolute association. Some in- PON1192 genotypes in epidemiological parathion or chlorpyrifos. The injected dividuals homozygous for PON1M55 studies (Richter and Furlong 1999). A enzyme also provided some protection have high PON1 levels and some homo- number of studies have been carried out against dermal exposure to the parent zygous for PON1L55 have low PON1 lev- attempting to relate the PON1 genotype compound chlorpyrifos in mice (Li et al. els, the opposite of what would be ex- with cardiovascular disease (for review, 1993, 1995). pected from statistical studies of popula- see Navab et al. 1996; Heinecke and Lu- What about the consequence of tions (Furlong et al., 2000; Brophy, V.H., sis 1998; Mackness et al. 1998). Unfor- having low PON1 levels? Recent experi- G.P. Jarvik, R.J. Richter, L.S. Rozek, G.D. tunately, virtually all of these studies ments with PON1 knockout mice gener- Schellenberg, and C.E. Furlong, in prep). have ignored the variability of PON1 ated by Dr. Jake Lusis (UCLA) and co- In earlier studies, we developed levels between individuals and have workers clearly demonstrated that low

PCR-based assays for determining PON1 considered only the PON1192 genotype PON1 levels resulted in a dramatic in- genotypes (Humbert et al. 1993), as well (Richter and Furlong 1999). The study crease in sensitivity to chlorpyrifos oxon as two-substrate activity assays that pro- by Haley et al. (1999) is therefore one of (Shih et al. 1998) and diazoxon (Furlong vided an accurate inference of the the first to examine the effects of both et al.). The availability of PON1 knock-

PON1192 genotype. Plotting rates of di- genotype and phenotype on possible en- out mice has provided a very informa- azoxon hydrolysis versus paraoxon hy- vironmental exposures. tive model system for examining sensi- drolysis for individuals in a population However, an important question to tivity to these toxins. It should be noted divided the population into three ask is “Do differences in rates of hydro- that the mutation that knocked out se- groups of individuals, individuals ho- lysis of substrates measured in vitro ac- rum PON1 also knocked out liver PON1, mozygous for PON1Q192, heterozygotes, tually reflect differences in sensitivity of leaving the mice totally devoid of PON1 and individuals homozygous for an individual to specific OP com- contribution to OP detoxication (Fur-

PON1R192 (Davies et al. 1996; Richter pounds?” The pathways for OP detoxi- long et al.). Surprisingly, the knockout mice did not demonstrate an increased sensitivity to paraoxon, the substrate for which PON1 was named. Thus, the in vivo evidence available to date indicates that low PON1 levels can result in increased sensitivity to specific OP compounds. The surprising ob- servation that PON1 knockout mice are not more sensitive to paraoxon points out the need to carry out exposure experiments in an appropriate in vivo model system. Because in vitro assays

have shown that the PON1R192 isoform does not hydrolyze sarin, or hydrolyzes it very slowly, it will also be important

to determine whether human PON1Q192 Figure 1 Pathways of OP detoxication. For the insecticides, a bioactivation reaction carried out by can provide in vivo protection against a cytochrome P-450 (mixed function oxidases) is required to generate the very toxic oxon forms that inactivate the cholinesterases (Murphy 1980). The nerve agents do not require bioactivation for sarin exposure. cholinesterase inhibition. In summary, while the studies re-

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Insight/Outlook ported by Haley et al. (1999) were car- Aldridge, W.N. 1953a. Biochem. J. 53: 110–117. Heinecke, J.W. and A.J. Lusis. 1998. Am. J. Hum. ried out with a small number of subjects, ———. 1953b. Biochem. J. 53: 117–124. Genet.62: 20–24 Blatter, M.C., R.W. James, S. Messmer, F. Barja, Humbert, R., D.A. Adler, C.M. Disteche, C. their conclusions are interesting. Gulf and D. Pometta. 1993. Eur. J. Biochem. Hassett, C.J. Omiecinski, and C.E. Furlong. War veterans were exposed to some 211: 871–879. 1993. Nat. Genet. 3: 73–76. compounds for which PON1 status Blatter Garin, M.C., R.W. James, P. Dussoix, H. Li, W.-F., L.G. Costa, and C.E. Furlong. Blanche, P. Rassa, P. Froguel, and J. Ruiz. 1997. 1993. J. Toxicol. Environ. Health 40: 337– (PON1192 genotype/phenotype) contrib- J. Clin. Invest. 99: 62–66. 346. utes to resistance, consistent with the Brealey, C.J., C.H. Walker, and B.C. Baldwin. Li, W.-F., C.E. Furlong, L.G. Costa. 1995. Toxicol. genetic and pharmacological data on 1980. Pestic. Sci. 11: 546–554. Lett. 76: 219–226. the PON1 polymorphism. However, Costa, L.G., R.J. Richter, S.D. Murphy, G.S. Mackness, M.I., S. Arrol, and P.N. Durrington. 192 Omenn, A.G. Motulsky, and C.E. Furlong. 1991. FEBS Lett. 286: 152–154. in a retrospective study of toxic expo- 1987. In Toxicology of pesticides: Experimental, Mackness, M.I., S. Arrol, C. Abbott, P.N. sures, it is difficult to determine the clinical, and regulatory Perspectives (ed. L.G. Durrington. 1993. Atherosclerosis 104: compounds to which individuals were Costa et al.), pp. 263–266. Springer-Verlag, 129–135. Heidelberg, Germany. Mackness, B., I. Michael, S.A. Mackness, W. exposed, as well as the level of expo- Costa, L.G., B.E. McDonald, S.D. Murphy, G.S. Turkie, P.N. Durrington. 1997. Br. J. Pharmacol. sure and the consequence of the expo- Omenn, R.J. Richter, A.G. Motulsky, and C.E. 122: 265–268. sure. Because an individual’s PON1 sta- Furlong. 1990. Toxicol. Appl. Pharmacol. Mackness, B., P.N. Durrington, and M.I. tus is stable over time, this parameter 103: 66–76. Mackness. 1998. Gen. Pharmacol.31: 329– Davies, H.G., R.J. Richter, M. Keifer, C.A. 336. that can be reliably determined at later Broomfield, J. Sowalla, and C.E. Furlong. 1996. Main, A.R. 1956. Can. J. Biochem. Physiol. times. Nat. Genet. 14: 334–336. 34: 197–216. In future OP poisoning cases, it will Eckerson, H.W., C.M., Wyte, and B.N. La Murphy, S.D. 1980. In Toxicology: The basic be important to encourage treating phy- Du. 1983. Am. J. Hum. Genet. 35: 1126– science of poisons, 2nd ed. (ed. J. Doull et al.), 1138. pp. 357–408. Macmillan Publishing Co., New sicians to obtain cholinesterase values Furlong, C.E., R.J. Richter, S.L. Seidel, L.G. Costa, York, NY. and to save frozen urine for analysis of and A.G. Motulsky. 1989. Anal. Biochem. Shih, D.M., L. Gu, Y.R. Xia, M. Navab, W.-F. Li, S. metabolites to use in later identification 180: 242–247 Hama, L.W. Castellani, C.E. Furlong, L.G. Furlong, C.E., W.-F. Li, V.H. Brophy, G.P. Jarvik, Costa, A.M. Fogelman, A.J. Lusis. 1998. Nature of toxicants and determination of levels R.J. Richter, D.M. Shih, A.J. Lusis, and L.G. 394: 284–287. of exposure. Where it is not feasible to Costa. 2000. NeuroToxicology Navab, M. J.A. Berliner, A.D. Watson, S.Y. Hama, obtain cholinesterase values in sus- Geldmacher-von Mallinckrodt, M., and T.L. M.C. Territo, A.J. Lusis, D.M. Shih, B.J. Van pected OP poisoning cases, it will still be Diepgen. 1988 Toxicol. Environ. Chem. Lenten, J.S. Frank, L.L. Demer, P.A. Edwards, 18: 79–196. and A.M. Fogelman. 1996. Aterioscler. Thromb. worthwhile to preserve frozen urine Haley, R.W., T.M. Kurt, and J. Hom. 1997. J. Am. Vasc. Biol.16: 831-842 samples for later analysis. Med. Assoc. 277: 215–222. Richter, R.J. and C.E. Furlong, 1999. Haley, R.W., S. Billecke, and B. La Du. 1999. Pharmacogenetics 9: 745–753. Toxicol. Appl. Pharmacol. 157: 227–233. Watson A.D., J.A. Berliner, S.Y. Hama, B.N. REFERENCES Hassett, C., R.J. Richter, R. Humbert, C. Chapline, La Du, K.F. Faull, A.F. Fogelman, M Adkins, S., K.N. Gan M., Mody, and B.N. La Du. J.W. Crabb, C.J. Omiecinski, and C.E. Furlong. Navab. 1995. J. Clin. Invest. 96: 2882– 1993. Am. J. Hum. Genet. 52: 599–568. 1991. Biochemistry 30: 10141–10149. 2891.

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PON1 Status and Neurologic Symptom Complexes in Gulf War Veterans

Clement E. Furlong

Genome Res. 2000 10: 153-155 Access the most recent version at doi:10.1101/gr.10.2.153

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