Reply

M. B. Abou-Donia1, L. B. Goldstein2 and W. A. Khan3

1 Departments of Pharmacology and Cancer Biology Duke University Medical Center, PO Box 3813 Durham, NC 27710 2 Department of Medicine (Neurology) Duke University Medical Center, PO Box 3813 Durham, NC 27710 3 Departments of Pharmacology and Cancer Biology Duke University Medical Center, PO Box 3813 Durham, NC 27710

To the Editor:

We appreciate the time and effort Dr. Schoenig invested in critically analyzing our work (Abou-Donia et al., 2001Go). Although he may reasonably have questions about details of our methodology, many of his points are unfounded.

Major deficiencies in our experiments cited by Dr. Schoenig begin with the number of control animals. We dispute this contention. As required by our Institutional Animal Care and Use Committee and the animal use protocol that was approved by the Department of Defense, we strive to use the fewest possible numbers of animals in our experiments that are sufficient to provide statistically valid results. In previous work, we found that control animals have very consistent performances on the sensorimotor tasks used in the current study. There was no justification for further increasing the number of control animals.

Dr. Schoenig incorrectly believed that the solvent (70% ethanol) was used to apply the dermally administered chemicals (DEET and permethrin) for the entire experiment whereas control rats received the vehicle for only the last 15 days. Control rats received daily dermal application of 70% ethanol for the entire experiment and water by gastric lavage for the last 15 days (controlling for oral administration of pyridostigmine bromide (PB).

Dr. Schoenig also claims that there were "deficiencies in the statistical treatment of the data including the fact that interactions were not analyzed, the degrees of freedom appear incorrect, inadequate adjustments were made for multiple comparisons, and none of the combination groups were directly compared to the control group." For the behavioral data, all of the analyses presented were based on appropriate multiple group comparison statistical tests (either two-way repeated measures ANOVA for parametric data or the Kruskal-Wallis test for nonparametric data). Post-hoc tests were applied (Fisher LSD for parametric data and Dunn’s Procedure for nonparametric data) and interactions of interest examined only if the overall test was significant. The degrees of freedom are correct as given. All of the behavioral analyses included a comparison of each group with the control group. Therefore, the analyses of the behavioral data appropriately accounted for the multiple comparisons dictated by the study design. We did not test every potential statistical interaction as they were not relevant to the questions under study.

Dr. Schoenig disputes the statement that, "each drug given alone had a significant behavioral effect, which tended to become more evident over time" and indicates that the PB data were "shown and discussed for the 30-day point of the experiment." Our experimental section clearly described that the treatment with PB was initiated on day 30 following the beginning with the treatment with DEET and permethrin. On day 30, the treatment with PB was carried out in the morning (8:30–9:00 A.M.) and the behavioral evaluations were carried out 3 h later. However, we never indicated that there was a statistically significant effect for PB (or any of the treatments) at the 30 day time point. These observations are consistent with findings of Hoy et al. (2000) that found significant drug interaction with PB and permethrin at 2 h following treatment. We first carried out a repeated measures ANOVA for each behavioral parameter encompassing all groups and both time-points. These statistically significant ANOVAs should be interpreted as indicating a difference between at least two of the groups for at least one time point for each behavioral test. Although the effects were not completely uniform across behavioral tasks, each treatment given alone resulted in a significant behavioral impairment. For all behavioral parameters except beam-walk time, there was also a significant treatment group x time interaction, with poorer performance at the second time-point. Hence, our summary statement is entirely appropriate and completely supported by the data. As indicated under Materials and Methods of our manuscript, page 306, "all behavioral testing was performed by a trained observer blind to the treatment status of the animal " and mentioning of "blindfolded" in the legend of Figure 1 and 2 was meant to reflect this fact only. However, the usage in the figure legend does not otherwise reflect on the experimental design, conduct or data analysis.

Dr. Schoenig also felt that the biochemical data were "inconsistent and confusing [and] coupled with inappropriate text interpretations." First, Dr. Schoenig notes that the expected inhibitory effect of PB on plasma cholinesterase was not observed. Indeed, PB treatment is well known to inhibit plasma cholinesterase activity and we did find a non-statistically significant ~10–15% inhibition in plasma cholinesterase activity. In view of the fact that PB is a reversible inhibitor of cholinesterase, and because of the short half-life of PB (~1.5 h), it is expected that the extent of inhibition after 24 h may not be pronounced as seen in our experiments. Second, Dr. Schoenig indicates that "inconsistencies in the findings defy any logical explanation," particularly with regard to the varying effects of DEET and permethrin on cholinesterase activity. We found that each chemical given alone caused an increased cholinesterase activity in one or more brain regions that was no longer observed when given together, but was associated with a decrease in activity in the brainstem. It could be speculated that the affected brain regions might be initiating an adaptive response to maintain the appropriate levels of acetylcholine required for proper synaptic functions. It is also possible, based on the data generated from transgenic mouse model overexpressing acetylcholinesterase (Beeri et al., 1997Go; Sternfeld et al., 1998Go), that prolonged increases in the enzyme activity might lead to neurodegeneration, thereby having secondary effects in some (i.e., brainstem) but not other brain regions. This explanation is consistent with our follow-up studies following subchronic (60 days) dermal application of DEET and permethrin to adult rats, alone or in combination, showing diffused neuronal cell death and cytoskeletal abnormalities in the cerebral cortex and the hippocampus, and Purkinje neuron loss in the cerebellum (Abdel-Rahman et al., 2001Go). Third, Dr. Schoenig paraphrases our statement that, "This inhibition [i.e., referring to combined exposure with PB and DEET/permethrin] in the brainstem may be mediated by PB ..." and remarked that, "PB was not administered in the set of experiments under discussion." However, our actual statement was taken out of context. Neither PB, DEET nor permethrin given alone significantly depressed brainstem cholinesterase activity, although there was a nonsignificant reduction in activity with PB and an increase in activity with DEET (Figure 3, top panel). However, PB + DEET and PB + permethrin significantly decreased cholinesterase activity in the brainstem (Figure 3, middle panel). This led to our speculation about the combined effect. We also observed a reduction in brainstem activity with DEET + Permethrin (Figure 3, middle panel). When exposure to DEET and permethrin occurs concurrently, an inhibitory response could occur through a variety of mechanisms. We chose not to speculate on these potential mechanisms because our study was not aimed at elucidating the mechanistic aspects of effects following combined exposure. Next, Dr. Schoenig disputes our suggestion that the three chemicals together could lead to added neurotoxic effects. Although treatment with PB, DEET and permethrin did not result in additive effects on cholinesterase activity in the brainstem as compared to the effect seen with two of the chemicals, significant inhibition of cholinesterase activity in the brainstem was observed when any of the three chemicals were given in combination.

Dr. Schoenig indicates that, "... none of the data for combination treatments are significantly different than for PB data alone," referring to brain choline acetyl transferase, m2 muscarinic and nicotinic acetylcholine receptor activity, and therefore that there is no support for the claim that, "The results suggest that exposure to these chemicals, alone or in combination, causes significant sensorimotor deficits and differential regulation of [these receptors] in the CNS." The data presented in Figure 5 show a significant increase in m2 muscarinic receptor ligand binding in the cortex following treatment with PB, DEET or permethrin, given alone or in combination. PB + DEET (Figure 6) resulted in a significant increase in nicotinic acetylcholine receptor activity in cortex. Thus, our statement reflects our finding that treatment with these chemicals alone or in combination causes differential regulation in the ligand binding densities of these receptors. The effects on sensorimotor function were reviewed above.

Our article was adequately referenced, however, we included relevant literature on the effects of DEET on the nervous system. Our results that dermally applied DEET caused sensorimotor performance deficit are consistent with the neurotoxicity of DEET in humans including death following extensive and repeated topical application (de Garbino and Laborde, 1983Go; Edwards and Johnson, 1987Go; Gryboski et al., 1961Go; Robbins and Cherniack, 1986Go; Roland et al., 1985Go; Zadikoff, 1979Go). The difference in exposures, behavioral tests and other portions of the experimental protocols could account for the difference between our findings and the earlier study of Dr. Schoenig and colleagues (Schoenig et al., 1993Go). We do not believe that Dr. Schoenig’s earlier study is a "benchmark," it merely provides data from different types of experiments than our study.

Dr. Schoenig disputes our statement that "exposure to physiologically relevant doses of PB, DEET and permethrin, alone or in combination, lead to sensorimotor deficits and alteration of the cholinergic system in rats." We believe that the data we presented clearly supports this conclusion with the doses used being physiologically relevant based on their observed effects.

Our study did not examine long-term behavioral consequences of transient exposure to these chemicals. However, we only suggested that exposure to these chemicals may have played a role in the long-term health consequences of exposure in Gulf War veterans. This statement is supported by our follow-up neuropathological studies (Abdel-Rahman et al, 2001Go).

We welcome discussion and comment on our report and recognize that clarifications of published experiments are often helpful. The use of differing methodologies helps to provide a fuller understanding of the potential health effects of putative toxicants. It is unfortunate that Dr. Schoenig felt the need to raise his concerns in such a harsh and negative manner, and to question the adequacy of Toxicological Sciences’ peer review process.

REFERENCES

Abdel-Rahman, A., Shetty, A. K., and Abou-Donia, M. B. (2001). Subchronic dermal application of N,N-Diethyl m-toluamide (DEET) and permethrin to adult rats, alone or in combination, causes diffuse neuronal cell death and cytoskeletal abnormalities in the cerebral cortex and hippocampus, and Purkinje neuron loss in the cerebellum. Exp. Neurol. 172, 153–171.[ISI][Medline]

Abou-Donia, M. B., Goldstein, L. B., Jones, K. H., Abdel-Rahman, A. A., Damodran, T. V., Dechkovskaia, A. M., Bullman, S. L., Amir, B. E., and Khan, W. A. (2001). Locomotor and sensorimotor performance deficit in rats following exposure to pyridostigmine bromide, DEET and permethrin, alone and in combination. Toxicol. Sci. 60, 305–314.[Abstract/Free Full Text]

Beeri, R., Le Novere, N., Mervis, R., Huberman, T., Grauer, E., Changeux, J. P, and Soreq, H. (1997). Enhanced hemicholinium binding and attenuated dendrite branching in cognitively impaired acetylcholinesterase-transgenic mice. J. Neurochem. 69, 2441–2451.[ISI][Medline]

de Garbino, J. P., and Laborde, A. (1983). Toxicity of an insect repellent: N,N diethyltoluamide. Vet. Human Toxicol. 25, 422–423.[ISI][Medline]

Edwards, D. L., and Johnson, C. E. (1987). Insect repellent-induced toxic encephalopathy in a child. Clin. Pharmacol. 6, 496–498.[ISI][Medline]

Gryboski, J., Weinstein, D, and Ordway, NK. (1961). Toxic encephalopathy apparently related to the use of an insect repellent. N. Eng. J. Med. 264, 289–291.[ISI]

Hoy, J. B., Cornell, J. A., Karlix, J. L., Schmidt, C. J., Tebbett, I. R., and van Haaren, F. (2000). Interactions of pyridostigmine bromide, DEET and permethrin alter locomotor behavior of rats. Vet. Human Toxicol. 42, 65–71.[ISI][Medline]

Robbins, P. J, and Cherniack, M. G. (1986). Review of the biodistribution and toxicity of the insect repellent N,N-diethyl m-toluamide (DEET). J. Toxicol. Environ. Health. 18, 503–525.[ISI][Medline]

Roland, E. H., Jan, J. E., and Rigg, J. M. (1985). Toxic encephalopathy in a child after brief exposure to insect repellent. Can. Med. Assoc. J. 132, 155–156.[Abstract]

Schoenig, G. P., Hartnagel, R. E., Jr., Shardein, J. L., and Vorhees, C. V. (1993). Neurotoxicity evaluation of N,N-diethyl-m-toluamide (DEET) in rats. Fundam. Appl. Toxicol. 21, 355–365.[ISI][Medline]

Sternfeld, M., Ming, G., Song, H., Sela, K., Timberg, R., Poo, M. and Soreq, H. (1998). Acetylcholinesterase enhances neurite growth and synapse development through alternative contributions of its hydrolytic capacity, core protein, and variable C termini. J. Neurosci. 18, 1240–1249.[Abstract/Free Full Text]

Zadikoff, C. M. (1979). Toxic encephalopathy associated with use of insect repellent. J. Pediatr. 95, 140–142.[ISI][Medline]





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