ELSEVIER Toxicology 102 (1995) 215 -220

Regulatory and research issues related to cholinesterase inhibition

Stephanie Padilla

Neurotoxicology Division (MD-74B). Health Effects Research L&oratory, U.S. Environmentd Protection Agency,

Research Triangle Park, NC 27711, USA

Abstract

Assessing the neurotoxic potential of organophosphate and carbamate pesticides should be greatly facilitated by the knowledge that the mechanism of action of these insecticides is presumed to be the inhibition of cholinesterase, the enzyme which controls the levels of neurotransmitter, acetycholine. Although the inhibition of cholinesterase activity is the recognized mechanism of action, many questions remain regarding the use of cholinesterase inhibition data as a critical effect for establishing risk of cholinesterase-inhibiting pesticides. Specifically, questions have arisen regarding whether blood cholinesterase inhibition correlates with inhibition in target tissues (e.g. brain or muscle) and whether cholinesterase inhibition in any tissue correlates with the adverse clinical and behavioral effects produced by exposure to cholinesterase-inhibiting pesticides. Studies in our laboratory indicate that blood cholinesterase inhibition in both acute and subchronic dosing regimens correlates with inhibition in other tissues, if measurements are taken at the appropriate times. Moreover, there is evidence in the literature and from our laboratory that cholinesterase inhibition correlates with the emergence and severity of clinical signs of poisoning by cholinesterase-inhibiting pesticides.

Keywords: Acetylcholinesterase; Cholinesterase; Organophosphate pesticides; Carbamate pesticides; Cholinesterase inhibitors; Cholinesterase inhibition, brain; Cholinesterase inhibition. blood; Cholinesterase inhibition, correlation; Cholinesterase inhibitors, risk assessment

1. Introduction

Knowing the mechanism of action of a toxicant or a class of toxicants should greatly facilitate the

*Corresponding author, Tel.: (919) 541-3956; Fax: (919) 54 I-4849.

risk assessment process. In the case of carbamate and organophosphate pesticides, the major mode of action is assumed to be the inhibition of an enzyme, acetylcholinesterase (E.C. 3.1.1.7).

The inhibition of acetylcholinesterase activity adversely affects animals because this enzyme nor- mally regulates the proper levels of the neuro- transmitter acetylcholine in the central nervous

Elsevier Science Ireland Ltd.

S.~DI n7nn-48?xmwmw-p

216 S. Pudillu I Toxicology 102 (1995) 215-220

system, the neuromuscular junction, the parasym- pathetic nervous system, the sympathetic synapses and the sympathetic innervation of the adrenal and sweat glands. If acetylcholinesterase activity is depressed due to exposure to a cholinesterase inhibitor, then the ordinarily rapid breakdown of released acetylcholine is slowed, resulting in over- stimulation of the target cells, manifested as a ‘cholinergic crisis’ which can be debilitating and even fatal (Ecobichon, 199 1; Marrs 1993). Al- though evidence has been accumulating which demonstrates that the actions of cholinesterdse inhibitors may be more diverse than that depicted above (Small, 1990; Eldefrawi et al., 1992; Layer, 1992; Massoulie et al., 1993) for simplicity, we will assume that the principal adverse effects of cholinesterase inhibitors are due to the inhibition of acetylcholinesterase activity.

In order to assess the risks of this class of compounds, the regulator must be able to proceed from data regarding the biochemical and behav- ioral effects to ultimately estimating levels of probable risk for humans. To make that progres- sion the regulator must decide how much weight should be assigned to a particular effect. Some of the usual endpoints or data available for consider- ation are the degree of inhibition of cholinesterase activity in the blood and brain and a description of clinical signs.

To be more precise, during the last 5 years, the U.S. Environmental Protection Agency has been grappling with the appropriate uses of cholinesterase inhibition data for risk assessment. Many pesticides are tested for neurotoxicity using protocols in which laboratory animals (usually rats) are administered the pesticide and blood samples are taken serially during exposure. Fol- lowing exposure, the animals are sacrificed and other tissues are collected for analyses of cholinesterase activity. Therefore, the resulting set of experimental data for regulatory review cus- tomarily includes chohnesterdse levels on many time-course blood samples and also cholinesterase levels in considerably fewer, other tissue samples (usually brain) only at time of sacrifice and possi- bly some rudimentary behavioral data (e.g. clini- cal signs of cholinergic overstimulation such as diarrhea or tremors). Of course, in human studies

or in accidental human exposure episodes, blood samples are the only tissue analyzed; clinical signs and symptoms may or may not be present as part of the data profile.

Cholinesterase inhibition in either the nervous system or muscle is accepted as an adverse effect because cholinesterase activity in these target tis- sues is known to participate in neurotransmission. Chohnesterdse activity in the blood however, ap- parently does not take part in neurotransmission and therefore, inhibition of circulating cholinesterase activity is typically classified only as a marker of exposure to organophosphate or carbamate compounds. Consequently, the risk as- sessor is faced with at least two very important questions: (1) does the level of cholinesterase inhi- bition in the blood reflect the inhibition in target tissues? and (2) do the levels of cholinesterase inhibition in the blood or target tissue correlate with alterations in the clinical or behavioral profile of the animal? These two questions are considered below.

2. Toxicological significance of blood cbolinesterase inhibition

One would think that after 50 years of intensive research that the literature on anticholinesterase toxicity would be replete with information on whether inhibition of circulating cholinesterase activity is related to cholinesterase inhibition in the nervous system or other target tissues such as skeletal muscle. In fact, very few studies exist that were specifically conducted to determine how well cholinesterase inhibition in blood mimics inhibi- tion in target tissues. Any relationship between blood cholinesterase activity and other tissues has, for the most part, been gleaned from studies not specifically designed to establish this relationship. In many of these studies, however, there does seem to be a correlation between blood cholinesterase inhibition and inhibition in target tissues (Miyamoto, 1969; Su et al., 1971; Koshakji et al., 1973; Reiter et al., 1973; Anand et al., 1977) even though other reports both in the clinical setting and the laboratory setting, have questioned this correlation (Karnik et al., 1970;

S. Padilla 1 Toxicology 102 (1995) 215-220 217

Endo et al. 1988; Jimmerson et al., 1989). Pope and coworkers have recently undertaken studies that show reliable correlations between blood and brain levels of cholinesterase activity in rats dosed with environmentally-relevant compounds. Their studies, however, were limited to either multiple dosages at one time-point (Pope and Chakraborti, 1992) or to one dose at multiple time-points (Pope et al., 1991).

Given the regulatory significance of this rela- tionship, research is needed to assess this correla- tion in a time- and dose-dependent manner. Both our laboratory and Pope’s laboratory have re- cently initiated these types of studies. As part of the poster session in this Symposium, Pope and co-workers have presented data showing excep- tionally high correlations between plasma cholinesterase inhibition and inhibition in other tissues such as heart, muscle and brain in both chlorpyrifos- and parathion-treated rats (Chauld- huri et al., 1993). In our laboratory, we have completed a detailed dose-response and time- course study of cholinesterase inhibition in the tissues of rats treated with various dosages of either chlorpyrifos or paraoxon (Padilla et al., 1994). These two compounds were chosen because although both are organophosphate compounds, they differ with regard to hepatic activation (chlorpyrifos requires hepatic activation; paraoxon does not) and time of peak effect (7 days for chlorpyrifos; 4 h for paraoxon). The results show strong correlations between blood, brain and muscle cholinesterase inhibition for both compounds at the time of peak cholinesterase inhibition - a time interval which also corresponds to the period when cholinesterase levels were most stable (i.e. l-3 weeks post-dosing for chlorpyrifos-treated rats and 4 h post-dosing for paraoxon-dosed rats). These data indicate that under appropriate condi- tions, the level of cholinesterase inhibition in blood may indeed, accurately reflect inhibition in target tissues.

treated repeatedly with a cholinesterase inhibitor. This is especially important since many of the tests conducted for regulatory purposes use a repeated-dosing regimen. Our laboratory has just completed a study which measured the cholinesterase activity in various tissues of rats treated weekly with chlorpyrifos (15, 30, or 60 mg/kg, s.c.) for 5 weeks. Animals were sacrificed and tissues collected during the exposure period (i.e. 1, 3, 5 weeks) and during the recovery period (i.e. 7 weeks) (Padilla, Wilson, Willig and Bush- nell, unpublished data). Cholinesterase activity, measured in whole blood and cortical hemi- spheres showed a strong correlation both during dosing and also during the recovery period (Fig. 1). Overall, however, the correlations were higher during the dosing period. That is, the relationship between blood cholinesterase activity and the ac- tivity in the target tissue is much more reliable

The data presented above were collected in animals dosed acutely with cholinesterase in- hibitors. It is not clear whether blood cholinesterase activity would also correlate with target tissue cholinesterase activity in animals

^ Whole 81006 Choll”crtero.e (a Control) Whole Blood Cholinesterole (I CO”,d)

~‘~~:, l;yyyf$-j $ 0% 20X 40% 601 BOX 100X ox 20% 40% 601 BOX ,00x

Whole Blood Chaline.tera*e (a Control) Whole 81OOd Chollnestsrole (a co”tro,)

Fig. I. Correlations between cholinesterase inhibition in whole

blood versus brain cholinesterase inhibition in animals treated

weekly with chlorpyrifos (0, 15, 30 or 60 mg/kg in peanut oil,

s.c.). Panels A,B,C show the correlation during the dosing: A,

after one dose; B, after two doses and C, after four doses.

Panel D shows the correlation between whole blood

cholinesterase levels and brain levels after 2 weeks of recovery.

Note that the correlation is very strong during the dosing

period, but begins to decrease in correspondence during the

recovery phase.

218 S. Padilla 1 Toxicology 102 (1995) 215-220

when the inhibition has reached steady-state than at times when the activity is recovering, a relation- ship also noted with acute dosing. For example, the data for whole blood cholinesterase inhibition vs. cortex inhibition are presented in Fig. 1. Dur- ing the course of dosing, the values for cholinesterase are falling (i.e. points are moving down and to the left) for both tissues, but there is a significant correlation between whole blood in- hibition and inhibition in the cortex at all time points. Even at tieek 7 when cholinesterase activ- ity is recovering, a correlation is still evident, although it is less than during the dosing period.

In summary, these preliminary data, both from our laboratory and others have shown that when the question of the correlation between blood and target tissue cholinesterase inhibition is investi- gated using both multiple dosage levels and tissue samples taken at the time of peak inhibition, there seems to be a predictable, strong relationship between inhibition in blood and inhibition in muscle or brain.

3. Correlation of behavioral measures with cholinesterase inhibition

Ideally, the regulator would like to base the toxicity profile of any cholinesterase inhibitor on signs and symptoms of poisoning. Even though the spectrum of signs (experimental animals and humans) and symptoms (reported by humans) associated with acute exposure to organophos- phate and carbamates are well-characterized, there is a paucity of information in the literature regarding the relationship between those alter- ations in behavior and the degree of cholinesterase inhibition in either blood or other tissues (recently reviewed in Annau 1992; D’Mello, 1992, 1993). This gap was recognized as a concern by members of the EPA sponsored Scientific Advisory Board/Scientific Advisory Pane1 which considered questions surrounding risk assessment of cholinesterase-inhibiting com- pounds: ‘Given the lengthy history of anti- cholinesterase agents, the lack of information on the correlation between central and peripheral nervous system effects and cholinesterase inhibi- tion is surprising’ (US EPA, 1990).

Close examination of the literature reveals that there are a few studies which examined the rela- tionship between tissue cholinesterase inhibition and behavioral alterations. In one often quoted study, Hoskins et al. (1986) found that the toxic signs elicited by nerve agents were not necessarily correlated with the degree of cholinesterase inhibi- tion in the striatum and that the signs were gener- ally not apparent until striatal cholinesterase inhibition exceeded 50%. In other studies, not cited as often, but seemingly more to the point since the investigators dosed animals with pesti- cides instead of nerve agents, significant behav- ioral changes were often noted at dosage levels which produced little (Hart, 1993) or no (Kurtz 1977; Roney et al., 1986; Dutta et al., 1992) cholinesterase inhibition.

For example, Roney et al. (1986) correlated brain and plasma cholinesterase inhibition with the taste aversions produced by three different organophosphates: diisopropylfluorophosphate (DFP), parathion or dichlorvos. It was noted that the relationship between brain cholinesterase inhi- bition and the taste aversion was linear for dichlorvos, but was sigmoidal for DFP and parathion, such that substantial decreases in sac- charin preference (i.e. increased taste aversion) occurred in the presence of no significant de- creases in brain cholinesterase activity (Fig. 5a in Roney et al., 1986). There was, however, a linear relationship between plasma cholinesterase inhibi- tion and taste aversion for all three organophos- phates (Fig. 5b in Roney et al. 1986), leading the authors to hypothesize that the inhibition of pe- ripheral, not central, cholinesterase activity was mediating the taste aversion in the organophos- phate-treated rats.

Recently we conducted a study in which we were able to determine how well brain cholinesterase inhibition correlated with the acute effects of paraoxon, the active metabolite of parathion (Padilla et al., 1992). The acute cholin- ergic effects were assessed using a Functional Ob- servational Battery (FOB) and an assessment of motor activity (Moser, 1989). Brain cholinesterase levels were measured in the same animals so that correlations could be established between behav- ioral changes and cholinesterase activity. The re-

Fig. 2. Correspondence between brain cholinesterase inhibition and three ditferent measures of behavioral change: A, motor activity; B, body temperature and C, gait score (all described in detail in Moser, 1989). Note that both body temperature (B) and gait (C) show a ‘threshold’ type of pattern with respect to brain chohnesterdse inhibition, i.e. there are significant de- creases in brain cholinesterase activity ( 5 50%) before changes in either body temperature or gait are apparent. In the case of motor activity (A), however, the relationship between the two endpoints is linear; animals with only 26% brain cholinesterase inhibition exhibit depressed motor activity. Figure adapted from Pddilla et al.. 1992.

suits indicated that some measures, such as gait change and body temperature, seemed to exhibit a threshold pattern relating brain cholinesterase in- hibition to changes in the measured endpoint (Fig. 2. panels B and C). Motor activity levels, however, appeared to have no threshold i.e. slight inhibition (26”/0) of brain cholinesterase activity was associated with modest, but significant, de- creases in motor activity, while more pronounced depression of brain cholinesterase activity resulted in larger decreases (Fig. 2 panel A). These results warrant further investigation and we are now planning studies to correlate several behavioral effects with cholinesterase inhibition in a variety of tissues in animals dosed with other, selected pesticides.

In summary, the review of the literature cou- pled with our recent data indicates that how well the degree of cholinesterase inhibition correlates with clinical or behavioral endpoints apparently depends both on behavioral endpoint (Hoskins et al. 1986; Padilla et al., 1992; Hart, 1993) and on the cholinesterase inhibitor (Roney et al., 1986;

102 (1995) 215-220 219

Hoskins et al. 1989). Therefore, knowing the mechanism of action of a group of chemicals certainly helps point the way in both the research and regulatory arenas, but it by no means answers all the questions.

Disclaimer

The research described in this article has been reviewed by the Health Effects Research Labora- tory, U.S. Environmental Protection Agency and approved for publication. Approval does not sig- nify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names and commercial products constitute endorsement of recommendation for use.

References

Anand, M., Khanna, R.N., Misra, D. and Sharma, H.K. (1977) Changes in brain acetylcholine of rats after dermal application of fenitrothion (Sumithion). Ind. J. Physiol. Pharmacol. 21 12lll24.

Annau, Z. (1992) Neurobehavioral effects of organophospho- rus compounds. In: J.E. Chambers and P.E. Levi (Eds) Organophosphates: Chemistry, Fate and Effects, Academic Press New York, pp. 419-432

Chauldhuri, J., Harp, P., Chakraborti, T. and Pope, C. (1993) Comparative dose-related effects of parathion and chlorpyri- fos on cholinergic markers and behavior. Presented at the 1993 HERL Symposium on Biological Mechanisms and Quantitative Risk Assessment.

D’Mello, G.D. (1992) Neurobehavioural toxicology of anti- cholinesterases. In: B. Ballantyne and T.C. Marrs (Eds) Clinical and Experimental Toxicology of Organophosphates and Carbamates, Butterworth-Heinemann, Oxford, pp. 61~ 74.

D’Mello, G.D. (1993) Behavioural toxicity of anti- cholinesterases in humans and animals - A review. Hum. Exp. Toxicol. 12, 3-7.

Dutta, H., Marcelino, J. and Richmonds, C. (1993) Brain acetylcholinesterase activity and optomotor behavior in bluegills, fzpomis macrochirus exposed to different concen- trations of diazinon. Arch. lnt. Physiol., Biochim. Biophys. 100. 331-334.

Ecobichon, D.J. (1991) Pesticides. In: M.O. Amdur, J. Doull and C.D. Klaassen (Eds), Casarett and Doull’s Toxicology: The Basic Science of Poisons, 4th edn., Pergamon Press, New York, p. 580.

Eldefrawi. A.T., Jett, D. and Eldefrawi, M.E. (1992) Direct actions of organophosphorus anticholinesterases on mus- carinic receptors. In: J.E. Chambers and P.E. Levi (Eds) Organophosphates: Chemistry, Fate and Effects, Academic Press, New York. pp. 257-270.

220 S. Pudillu I TosicologJ 102 (1995) 215-220

Endo, G., Horiguchi, S., Kiyota, 1. and Kurosawa, K. (1988) Serum cholinesterase and erythrocyte acetylcholinesterase activities in workers occupationally exposed to organophos- phates. Proc. ICMR Sem. 8, 56ll564.

Hart, A.D.M. (1993) Relationship between behavior and the inhibition of acetylcholinesterase in birds exposed to organophosphate pesticides. Environ. Toxicol. Chem. 12, 321-336.

Hoskins, B., Fernando, J.R., Dulaney, M.D., Lim, D.K., Liu D.D., Watanabe, H.K. and Ho, I.K. (1986) Relationship between the neurotoxicities of soman, sarin and tabun and acetylcholinesterase inhibition. Toxicol. Lett. 30, I21 - 129.

Jimmerson, V.R., Shih, T.M. and Mailman, R.B. (1989) Vari- ability of soman toxicity in the rat: Correlation of biochem- ical and behavioral measures. Toxicology 57, 241-254.

Karnik, V.M., Ichaporia, R.N. and Wadia, R.S. (1970) Cholinesterase levels in diazinon poisoning. I. Relation to severity of poisoning. J. Assoc. Physicians of India 18, 482-487.

Koshakji, R.P., Cole, J. and Harbison, R.D. (1973) Influence of injection volume on parathion toxicity and plasma and brain cholinesterase inhibition. Res. Commun. Chem. Pathol. Pharmacol. 6, 677-687.

Kurtz, P.J. (1977) Behavioral and biochemical effects of the carbamate insecticide, Mobam. Pharmacol. Biochem. Be- hav. 6 303-310.

Layer, P.G. (1992) Towards a functional analysis of cholinesterases in neurogenesis: Histological, molecular and regulatory features of BChE from chicken brain. In: A. ShalTerman and B. Velan (Eds), Multidisciplinary Ap- proaches to Cholinesterase Functions, Plenum Press, New York, pp. 223-231.

Marrs, T.C. (1993) Organophosphate Poisoning. Pharm. Ther. 5, 51-66.

Massoulie, J., Pezzementi, L., Bon, S., Krejci, E. and Vallette, F.M. (1993) Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 3 I -91.

Miyamoto, J. (1969) Mechanism of low toxicity of Sumithion toward mammals. Res. Rev. 25. 251-264.

Moser, V.C. (1989) Screening approaches to neurotoxicity: A functional observational battery. J. Am. Coil. Toxicol. 8 85-93.

Padilla, S.. Moser. V.C., Pope, C.N. and Brimijoin. W.S. (1992) Paraoxon toxicity is not potentiated by prior reduc- tion in blood acetylcholinesterase. Toxicol. Appl. Pharma- col. 117, 110~115.

Padilla, S., Wilson, V.Z. and Bushnell. P.J. (1994) Studies on the correlation between blood cholinesterase inhibition and ‘target tissue’ inhibition in pesticide-treated rats. Toxicology 92, II -25.

Pope, C.N. and Chakraborti, T.K. (1992) Dose-related inhibi- tion of brain and plasma cholinesterase in neonatal and adult rats following sublethal organophosphate exposures. Toxicology 73, 35-43.

Pope, C.N., Chakraborti, T.K., Chapman, M.L.. Farrar J.D. and Arthun, D. (1991) Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophos- phorothioate insecticides. Toxicology 68. 51-61.

Reiter, L., Talens, G. and Woolley, D. (1973) Acute and subacute parathion treatment: Effects on cholinesterase ac- tivities and learning in mice. Toxicol. Appl. Pharmacol. 25. 582-588.

Roney, P.L., Costa, L.G. and Murphy, SD. (1986) Condi- tioned taste aversion induced by organophosphate com- pounds in rats. Pharmacol. Biochem. Behav. 24, 737-742.

Small, D.H. (1990) Non-cholinergic actions of acetyl- cholinesterases: Proteases regulating cell growth and devel- opment? Trends Biol. Sci. 15, 2133216.

Su, M.Q., Kinoshita, F.K., Frawley, J.P. and Dubois, K.P. (1971) Comparative inhibition of aliesterases and cholinesterase in rats fed eighteen organophosphorus insecti- cides. Toxicol. Appl. Pharmacol. 20, 241-249.

US EPA (1990) U.S. Environmental Protection Agency Re- port of the SAB/SAP Joint Study Group on Cholinesterase: Review of Cholinesterase Inhibition and Its Effects EPA- SAB-EC-90-014.