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(Published in: Archives of Environmental Health, Vol 48, pp. 89-93, 1993
IMMUNOLOGIC ABNORMALITIES IN HUMANS EXPOSED TO CHLORPYRIFOS: PRELIMINARY OBSERVATIONS Jack D. Thrasher, Ph.D., Roberta Madison, Dr.P.H. Alan Broughton, M.D., Ph.D.
exposure to determine changes in the peripheral immune system. The subjects were found to have a high rate of atopy and antibiotic sensitivities, elevated CD26 cells (p <0.01), and a higher rate of autoimmunity, compared to with two control groups. Autoantibodies were directed toward smooth muscle, parietal cells, brush boarder, thyroid gland, myelin and ANA. Chlorpyrifos exposure was implicated in the immunologic abnormalities reported. The immunologic changes were similar to those reported for other pesticides.
INSECTIDE EXPOSURE is increasingly implicated in peripheral blood immunologic abnormalities in humans. For example, pentachlorphenol (PCP)-exposed subjects have activated T cells (i.e. CD25, CD26) autoimmunity, function immunosuppression, and B cell dysregulation.1 Exposure to chlordane/heptachlor leads to T cell activation (CD26), increased cortical lymphocytes (CD1), decreased frequency of suppressor/ inducer phenotype (CD45RA/T4), lowered mitogen response, and autoimmunity.2, 3 Some individuals, following dioxin exposure (a contaminant of chlorinated pesticides), have a depressed delayed-type hypersensitivity reaction, decreased T4 and T11 cells, and a suspected increase in cortical thymocytes.4 Moreover, women who are exposed to aldicarb in drinking water have an elevated Candida antigen stimulation reaction, increased T8 cells, and a decreased T4/T8 ratio.5 In this paper, we present preliminary observations on changes in peripheral blood immunolgic phenotype frequencies and report on the presence of autoantibodies after exposure to chlorpyrifos. Materials and Methods
Patients and controls. Twelve patients (4 males and 8 females, 34 +8 y of age) were referred to the laboratory by their physicians for immunologic testing because of chronic health complaints that were unrelated to previous health histories (Table 1). Pesticide exposures were confirmed through physician records, subject interviews, material safety data sheets, and laboratory analyses. The exposures were as follows: toxic spill (n = 1), office (n = 2), bus (n = 1), classroom ( n = 1), and home (n = 7). Except for the spill, all insecticides were applied by licensed operators. Measurements of pesticide residues were not made except in two cases. Surface wipes at 5 mo post application showed residue levels of chlorpyrifos from <10 to 2,900 ppm. Two groups of controls were used. Group A consisted of 28 student volunteers (15 males, 13 females), average age 29 +9 y, who were exposed to formaldehyde as described previously.7 Groups B consisted of 29 healthy home dwellers (13 males, 16 females), average age 54 +14 y, also as described previously.8 The atopy history (hay fever, asthma) of Group A was unknown, where as that of Group B was reported previously. Blood collection. Venous blood samples were drawn from the individuals during the 1-4.5 y that followed exposure. All samples, including those obtained from controls, were collected under the supervision of an attending physician in silicone-treated sodium heparinized glass evac-tubes. The blood samples were transported to the laboratory and were used within 24 h with a cell viability of 90% or greater (trypan blue exclusion). All samples were assigned a computer-generated accession number. Quality assurance was performed by positive and negative unkown samples (autoimmunity). Lymphocyte markers. The total peripheral white blood cell (WBC) and lymphocyte counts were performed with a Coulter T-540 counter (Coulter. FL). Lymphocyte marker procedures are described elsewhere.7, 9 In brief, peripheral mononuclear cells were isolated by Ficoll Hypaque density gradient.10 The percentages and absolute numbers (ABS) of lymphocyte subsets per centimeter of blood were enumerated by fluorescent microscopy, utilizing antibodies to surface markers as follows: CD5 (LEU1), T cells); CD4 (LEU3A, The helper cells); CD8 (LEU2A, T suppressor cells); CD19 (LEU10, B cells) [Beckton-Dickinson, Los Angeles, CA); CD25 (IL2 receptor positive cells); and CD25 (TA1 positive cells [Coulter, FL]). All surface markers, except CD26, were identified by indirect immunofluorescence. The CD26 cells were determined by a direct immunofluorescent method.11, 12 Autoantibody determinations. Antismooth muscle (ASM), antiparietal cell (APC), antibrush border (ABB),antimitochondrial (AMIT) and antinuclear (ANA) antibodies were detected by an indirect immunofluorescent method and were expressed positive at a titer of 1:20.13
Antimyelin antibodies were determined by an indirect immunofluorescent method, and rabbit sciatic nerve replaced cervical cord.14 Titers were expressed positive at 1:8. Autoantibodies to thyroglobulin and thyroid peroxidase (microsomal) were determined by the method of Beever et al15 and distributed by Kronus (San Juan Capistrano, CA). One subject had an ELISA procedure performed (Immunosciences Lab, Los Angeles, CA). Results Health complaints, atopy, and smoking histories. The exposed subjects expressed multiple-organ symptoms for which they sought medical attention. These included an initial flu-like illness followed by chronic complaints of fatigue; central nervous system problems (headaches, dizziness, loss of memory); upper and lower respiratory symptoms; joint and muscle pain; and gastrointestinal disturbances. In addition, individuals who were in environments that contained low concentrations of chemicals, particularly chlorpyrifos and other organophosporus insecticides, experienced symptoms. Treating physicians reported that the chronic health problems were unrelated to the patients' atopy. Table 1 - Summary of Characteristics of 12 Patients Exposed to Chlorpyrifos
*1 = hay fever, 2 = childhood asthma, 3 = molds, 4 = bronchitis, 5 = penicillin, 6 = tetracycline, and 7 = sulfa drug Of the 10 individuals for whom health histories were obtained, the following was found: 8 had seasonal hay fever, 1 had asthma as a child, 1 had mold sensitivities, 1 ( a smoker) had bronchitis, and 5 had sensitivities to antibiotics. One individual developed multiple allergies and 4 antibiotic sensitivities after exposure. After the exposure, two females were diagnosed with SLE (teacher) and a lupus-like syndrome by a treating physician (Table 1). Peripheral blood cell counts. The ABS was elevated for WNC, total lymphocytes, CD5, CD4, CD8 and CD26 cells, compared to the controls (Table 2). Analysis of variance showed that only the CD26 cells were significantly different (p <.01). The percentage of CD cells, p <.05) and CD4 (p <.05>.01) was significantly decreased. In contrast, the percentage of CD26 cells was greatly elevated (p <.01), compared to the controls. The CD25 cells were decreased, but not significantly, whereas CD19 cells were unchanged, compared with controls. Table 2. - Mean Absolute Counts (ABS) of WBC, lymphocytes, and Lymphocyte Subsets and Mean Percentage of Lymphocyte Subsets +SDs for Exposed and Two Controls (Groups A and B)
Autoantibodies. All autoantibodies(except antimitochondrial) were elevated, compared with individuals from both control groups (Table 3). The odds ratio for ABB was significant (95% confidence interval (95% CI). Also, the odds ratios for the percentage of individuals with 1 and 2 or more autoantibodies were significant (95% CI), whereas that for 3 or more was not. Four of the 9 (44.4%) chlorpyrifos subjects were positive for antimeylin antibodies (data not shown). In comparison, 24 % of Group A was positive.
Five subjects were tested for antithyroid antibodies. Three subjects were positive for antimichrosomal; one had antithyroglobulin, and two were negative for both autoantibodies (Table 4). Discussion These data demonstrate several immunologic abnormalities: (a) history of atopy and antibiotic sensitivities, (b) decreased percentage of T cells, (c) increased CD26 (Ta1) cells, and (d) autoimmunity. Each abnormality will be discussed briefly. Table 3. - Percentage of Each Autoantibody Found in Sera of Exposed (EXP) versus Control Groups A and B (upper part of table) and Percentage of Individuals Who Had 1 or More, 2 or More, or 3 autoantibodies (lower part of the table).
Note: Odds ratios (95 % CI) are for exposed compared with control groups A and B.
The presence of atopy and antibiotic sensitivities suggests that the subjects in this study may be predisposed to chemical injury. For example, pencillins and penicilliamine are associated with allergies and autoimmunity.17 Although this is a small and possibly nonrepresentative sample, these observations suggest that HLA phenotypes (e.g., DR3, DR4, and DR5) and atopy may play a role in some forms of chemical hypersensitivities and should, therefore, be studied further.17 Given that the majority of the subjects in this study had a previous history of hay fever and antibiotic sensitivity, it may be argued that the observed immunologic abnormalities are related to this history. Certain observations, however, contradict this argument. First, one patient did not have such a history, but developed multiple allergies and antibiotic sensitivities after exposure. Second, four of the subjects developed antibiotic sensitivities after the insecticide exposure. Third, two patients were diagnosed with either SLE or lupus-like syndrome after their exposure. And, finally, there were nine additional individuals who were exposed to chlorpyrifos who had identical immunologic findings. Three of the nine subjects did not have prior histories of sensitivities, but have developed them subsequent to exposure (work in progress). The findings for WBC, lymphocytes, and lymphocyte subsets, although not statistically significant, showed an increase in absolute numbers, compared with the control groups. Interestingly, the percentage of CD5 and CD4 cells were reduced significantly, probably resulting from the many CD26 cells. Similar observations have been reported following exposure to PBBs,19 PCBs,20 and aldicarb.5 The exact nature of the T and B cell changes in this and other toxic exposures is not known. However, the changes are compatible with an increase in cortical thymocytes and CD10 cells and with decreased suppressor/inducer cells, all of which are reported for chlordane3 and PCP1 exposure. Table 4. -Summary of Autoantibodies to Thyroglobulin and Thyroid Peroxidase in the Five Subjects Tested.*
a = Radioimmunassay
Increased expression of CD26 (TA1) cells, associated with autoimmunity, is inferred from observations in multiple sclerosis,20 and following exposure to formaldehyde,7,8 chlordane/heptachlor,2,3 and PCP.1 Exposure to chlorpyrifos adds to and extends previous observations on xenobiotics. Therefore, the findings suggest that chlorpyrifos exposure is casually related to CD26 expression and autoimmunity. It is not known at this time what the mechanisms are regarding xenobiotic exposure and subsequent immunologic abnormalities, e.g. changes in T and B cell phenotypes and expression and the presence of autoimmunity. However, the observations regarding antibiotic sensitivities - both before and after exposure - point to a possible roll of HLA phenotypes. In addition, as suggested by McConnachie and Zahalsky,3 it is possible that continuous release of metabolites stored in adipose tissue may also play a role in these immunological abnormalities. This is intriguing because the chlorinated metabolites of chlorpyrifos are stored in adipose tissue.21 In conclusion, the presence of several different types of autoantiboides, e.g. antimyelin, antismooth muscle, antibrush boarder, and antimicrosomal, indicates that generalized tissue injury has occurred. Moreover, these identical observations have been made in additional chlorpyrifos patients (research in progress). Thus, chlorpyrifos as used in pesticide sprays, should be examined more closely as a probable immunotoxin. We thank Gilbert Salazar and the technical staff for their assistance. We deeply appreciate the considerate attention provided by the physicians: David Imes, Donald McNeil, Alan Robbins, David Busher, Fred A. Hurst, Paula Davey, George R. Borrell , and Linwood W. Custalow. Request for reprints should be sent to Jack D. Thrasher, Ph.D., International Institute of Research for Chemical Hypersensitivity, 11330 Quail Creek Road, Northridge, CA 91326. References 1. McConnachie PR, Zahalsky A. Immunological consequences of exposure to pentachlorophenol. Arch Environ Health 1991; 46:249-55. 2. Broughton A, Thrasher JD, Madison RA. Chronic health effects and immunological alteration associated with exposure to pesticides. Comments Toxicol 1990; 4:59-71. 3. McConnachie PR, Zahalksy A. Immune alterations in humans exposed to the termiticide technical chlordane. Arch Environ Health 1992; 47(4)295-301. 4. Knutsen AP, Rodman St, Evans RB, et al. Immune studies in dioxin-exposed Missouri residents: Quail Run. Bull Environ Contam Toxicol 1987; 39:481-89 5. Fiore MC, Anderson HA, Hong R. Chronic exposure to aldicarb-contaminated groundwater and human immune function. Environ Res 1985; 41:633-45. 6. Rosenthal NE, Cameron CL. Exaggerated sensitivity to an organophosphate pesticide. Am J Pyschiat 1991; 148:2. 7. Thrasher JD, Broughton A, Madison RA. Immune activation and autoantibodies in humans with long-term inhalation exposure to formaldehyde. Arch Environ Health 1990;45:217-23. 8. Madison RA, Broughton A, Thrasher JD. Immunologic biomarkers associated with exposure to exothermic by-products of a urea formaldehyde spill. Environ Health Perspect 1991; 94:219-23. 9. Broughton A, Thrasher JD. Antibodies and altered cell-mediated immunity in formaldehyde-exposed humans. Comments Toxicol 1988; 2:155-70. 10. Boyuma A. Isolation of mononuclear cells and granulocytes from blood. Scan J Clin Lab Invest 1968; 21(suppl 92):77-89. 11. Englemane EG, Warnke R, Fox FI, Levy R. Studies on human lymphocyte T-antigen recognized by monoclonal antibody. PNAS 1981: 78:91-95. 12. Fox DA, Hussey RE, Fitzgerald KA, et al. TA1, a novel 105 KD human T cell activation marker defined by a monoclonal antibody. J Immunol 1984: 133:351-54. 13. Nakamura RM, Tucker ES. Antibodies as reagent. In Henry JD (Ed): Diagnosis and management by laboratory methods. Philadelphia:PA. WB Saunders, 1982; pp. 122-27. 14. Edgington TS, Dalessio DJ. The assessment by immunofluorescence methods of human antimyelin antibodies in man. J Immunol 1970; 105:248-55. 15. Beever K, Bradbvury J, Phillips D, et al. Highly sensitive assays of autoantibodies to thyroglobulin and thyroid peroxidase. Clin Chem 1989; 39:1949. 16. Holford TR, Walter SD, Dunnet DW. Simultaneous interval estimates of the odds ratio in studies with two or more comparisons. J Clin Epidemiol 1989; 42:427-24. 17. Gleichman E, Kimel I, Purchase IFH. Immunotoxicology: suppressive and stimulatory effects of drugs and environmental chemicals on the immune system. Arch Toxicol 1989; 63:257-73. 18. Lu YC, Wu YC. Clinical findings and immunological abnormalities in Yu-Chen patients. Environ Health Perpsect 1985; 59:27-29 19. Lee T-P, Chang K-J. Health effects of polychlorinated biphenyls. In Dean J, et al (Eds): Immunotoxicology and Immunopharmacology. New York:Raven Press, 1985; pp. 415-22. 20. Hafler DA, Fox A, Manning ME, et al. In vivo activated lymphocytes in the peripheral blood and cerebrospinal fluid in patients with multiple sclerosis. New Engl J Med 1985; 312:1405-11. 21. Dishburger HJ, McKellar RL, Pennington JY, Rice JR. Determination of residues of chlorpyrifos, its oxygen analogue, and 2,5,6-trichloro-2-pyridinol in tissues of cattle fed chlorpyrifos. J Agricul Food chem. 1977; 25:1325-29. |
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