(Information on Concentrations of Chlordane in two of the homes is appended at the end of Discussion)

Patients with multiple subjective health complaints associated with confirmed inhalation exposure to chlordane, malathion and Dursban were examined for signs of immune activation and the presence of autoantibodies. Elevated CD26 (Ta1) cells and a high frequency of autoantibodies were found. These subjects have an activated immune system as determined by the presence of CD26 cells and autoantibodies. It appears that exposure to pesticides leads to chronic symptomolgy that is associated with autoantibodies and elevated CD 26 cells.

Key words: chlordane, dursban, malathion, immune activation, autoantibodies

INTRODUCTION

A pesticide is a biocide (i.e. it kills life). Information on toxicity is obtained from toxicological tests performed on target organisms, laboratory species (nonmammalian and mammalian), and acute human exposure. However, when it comes to long-term health effects on humans, little or no information is available. Thus, human exposure to a pesticide is examined from acute symptomology, while chronic health effects are either ignored or accepted as not related to the exposure. In this communication we will present the results of immunological studies on patients with chronic health complaints following exposure to chlordane and organophosphates (malathnion, Dursban.

MATERIALS AND METHODS

Lymphocyte Surface Markers

All procedures were performed on heparinized venous blood within 24 hours following collection. The total peripheral white cell count (WBC) was performed using a Model F Counter (Coulter, FL). The total lymphocyte count was done by blood smear examination. Lymphocyte surface marker procedures are published elsewhere.¹ In brief, peripheral mononuclear cells were isolated using Ficoll Hypaque gradient.² The percentages and absolute numbers (ABS) of lymphocyte subsets per mm³ of blood were determined utilizing monoclonal antibodies to surface markers: CD5 (LEU1); CD4 (LEU3A); CD8 (LEU2A; and CD19 (LEU10) {Becton Dickinson,

Los Angeles}; and CD26 (Ta1 positive) and CD25 (IL2 receptor positive) {Coulter, FL}. All surface markers, except CD26, were identified by indirect immunofluorescence.³ CD26 cells were determined by a direct immunofluorescent method.⁴

Autoantibody Screen

Antismooth muscle (ASM), antiparietal cell (APC), antibrush boarder (ABB), antimitochondrial (AMIT) and antinuclear (ANA) antibodies in the subject's and control sera were detected by an indirect immunofluorescent technique.⁵ Autoantibodies were expressed as positive at 1:20.

Antimyelin Autoantibodies

Antimyelin autoantibodies were done by indirect immunofluorescence as described by others, and, whereby, rabbit sciatic nerve replaced cervical cord.⁶ Titers were expressed as positive at a dilution of 1:8.

Statistical Analysis

T tests were done to examine whether a difference existed in the means (absolute and percentage) for WBC, lymphocytes, lymphocyte subsets, and helper/suppressor ratios. Odd ratios were calculated to determine whether the patients were at a higher risk of autoantibodies (ASM, APC, ABB, AMIT and ANA) than the controls.

CHLORDANE -- INTRODUCTORY COMMENTS

Chordane and its isomers are metabolized via mixed function oxidase (cytochrome P450) and glutathione S-transferase system.^7,8 The major metabolites, trans-nonachlor, oxychlordane and heptachlor epoxide are readily detected in the blood of pesticide applicators.⁹ Chlordane, heptachor, and heptachlor epoxide are carcinogenic in rodents, causing hepatic and extrahepatic tumors.^10,11 Evidence for carcinogenicity in humans rests upon case histories. Exposure to the pesticide is associated with neuroblastoma¹² and myelomonocytic, myeloid and lymphatic leukemias.^13-15 Other blood dyscrasias include aplastic anemia, megaloblastic anemia, pernicious anemia, idiopathic thrombochytopenia purpura, and hemolytic anemia.^12-16 One of the known causes for the latter three dyscrasias is autoimmunity.¹⁷ In this section we report the presence of elevated CD26 (Ta1) cells and a high incidence of autoantibodies in 24 subjects with chronic health complaints following chlordane/heptachlor application in their homes for termite infestations.

CHLORDANE PATIENTS AND COMPARISON GROUP

There were 24 patients (9 females, 15 males, average age 29+15.4 yrs) from seven families who comprised this study. All were exposed to chlordane used as a termiticide for periods ranging from 4 months to 4 years. None of the homes are currently occupied and one had to be razed. Ambient concentrations of chlordane were examined by both surface wipes and air sampling. Although the data were not available at this writing, all homes were positive for chlordane/heptachlor residues.

The comparison group was composed of students (15 male, 13 females, mean age = 29+9 yrs) exposed to formaldehyde for 13 hours per week for 28 weeks in a classroom.18 The students stated adverse effects during exposure were eye, nose, and throat irritation with a pungent odor of formaldehyde. Immunologic tests were performed one year after the last classroom exposure to formaldehyde. The students were asymptomatic at the time of testing.

Chlordane Symptoms

Each of the subjects had multiple organ symptomology that has continued since cessation of exposure to the termiticide. The symptoms followed and initial flu-like illness and include the following: fatigue, malaise, central nervous system (headaches, loss of memory, difficulty with task completion, disorientation or dizziness), G.I. tract (nausea, diarrhea, lower bowel cramping), upper and lower respiratory tract (rhinitis, sinusitis, bronchitis, shortness of breath, frequent infections) skin rash, muscle and joint pain, and peripheral neuropathy (pain and tingling of finger tips, tremor). Some patients expressed an olfactory sensitivity to environments containing low concentrations of chemicals (e.g. new carpets, diesel exhaust, fresh paints, perfumes), which precipitated CNS and respiratory tract symptoms.

One of the subjects (male: age, 11 yrs) was diagnosed with a malignant pheocytochroma with elevated catecholamines at one year following the initial chlordane exposure. A husband and wife were diagnosed with Guillian-Barrie (G-B) syndrome following chlordane exposure. The remaining subjects, although with multiple symptoms, had negative diagnostics for the continuing health problems. The immunologic tests were performed between 2 to 4 years following cessation of exposure to chlordane/heptachlor.

CHLORDANE RESULTS

Absolute Counts and Percentages of Peripheral T cells

The total white cell, lymphocyte, and T cell (CD5, CD4, CD8) counts, the percentage of T cells and T (CD4/CD8) ration for controls and the chlordane exposed subjects are listed in Table I. No difference was observed in the mean absolute counts and percentages of each type (WBC, CD5, CD4, CD8) between the comparison group and the patients. Also, no difference in the CD4/CD8 ratio was observed.

The Absolute Count and Percentages of CD26, CD25 and CD19 Cells

As shown in Table II the CD26 cells were significantly different (p = <0.001) in the mean absolute numbers and percentages in the chlordane groups (639+584 cell/mm³; 23+18 %) versus the students (122+93 cells/mm³; 5.1+3.6%). No difference between the comparison group and the chlordane subjects was found for CD25 cells. On the other hand, the absolute counts for CD19 were higher (p = >.05<.01) in the chlordane group (277+208 cells/mm³) as compared to the students (143+103 cell/mm³), while no difference was observed in the percentage of CD19 cells.

The Precentage of Autoantibodies

The percentage of each autoantibody detected at a dilution of 1:20 in the sera of chlordane and comparison subjects are represented in Table III. The percentage of each autoantibody was consistently higher in the chlordane group. The most frequent autoantibody was ASM (75%), followed by APC (70%), ABB (50%) ANA (35%) and AMIT (5%). When the rate at which autoantibodies (presence of an autoantibody) was determined, the chlordane subjects were higher than the comparison group, Table III.

Odds ratios for both the percentage of each autoantibody and the occurrence of autoantibodies were performed comparing the two groups. The odds ratio for 4 of the autoantibodies were as follows: ASM (18), APC (63), ABB (13) and ANA (14.5). Three of the odds ratios were significant at p = <0.01, while 1 was significant at p = <0.05. Moreover, the rate of occurrence of autoantibodies (i.e. one or more, two or more, three or more) was greater in the chlordane subjects as compared to the students (p = <0.05).

Table I. This table presents the mean absolute numbers of WBCs, lymphocytes, and the percentage of T cells with H/S ratio in the peripheral blood of the students and patients. The absolute numbers (ABS) are in cells/mm^3.

Table II. This table presents absolute numbers, the percentages, and t- and p values for CD26, CD25, CD19 in controls and chlordane subjects. The absolute numbers (ABS) are in cells/mm.3

Table III. This table presents the percentage of controls and chlordane subjects with each autoantibody. The upper portion of the table presents the percentage of each autoantibody that was found, while the lower portion gives the percentage at which one, two, three, or four or more autoantibodies were observed.

Antimyelin Autoantibodies

Antimyelin autoantibodies were not detected in any of the comparison subjects at a dilution of 1:8. Nine of the eleven (81.8 %) chlordane patients were positive at 1:8

ORGANOPHOSPHATES -- INTRODUCTORY REMARKS

At the present time we have examined three individuals exposed to organophosphates. Two had severe over exposure to dursban (chlorpyrifos) and one to malathion. Dursban [0,0-dimethyl 0-(2,4,5-trichloropyridinol)-phosphorthionate] is classified as an organophosphate, although three chlorides are attaches to the pyridinol ring. Dursban, like malathion [0,0-dimethyl-S-(2,2-dicarboethoxyethyl) dithiophosphate], inhibits cholinesterase activity by permanently deactivating the enzyme. Consequently, the liver must synthesize new enzyme before plasma levels can be restored, while the bone marrow must produce new RBCs with an active enzyme before cholinesterase activity, is restored to pre-exposure levels.¹⁹ Both pesticides are detoxified by cytochrome P450 mixed oxidases as well as GSH transferase. The primary metabolites of dursban are glucoruonides 5,6-trichloro-2pyridinol, 3,5,6-trichloro-2-pyridinol, and 3,5,6-trichloro-2-pyridinol glucoside.¹⁹ The major metabolites of malathion are malaoxon (an epoxide) and phosphoric, thiophosphoric and dithiophosphoric acid derivatives^.20

ORGANOPHOSPHATGE PATIENTS

Patient 1: A 40-year old female, life time smoker with an unremarkable health history. At the age of 37 her place of business (pet shop) was given an application of Dursban/Vapona. In the following days fish and birds died. Her health problems and immunolgic findings are presented in Table IV.

Patient 2: A 33- year old male, life time nonsmoker with an unremarkable health history. He worked as a manager at a cattle ranch. His first exposure to Dursban occurred at age 28 when the pesticide was used on cattle. The exposure continued for the next 14 months at which time he had a massive exposure resulting from a spill. Protective clothing, rubber gloves, or a respirator were not used during the exposures. His symptomology and immunologics findings are presented in Table IV.

Patient 3: a 62-year old female with a history of asthma, arthritis and hypothyroidism was exposed to malathion at the age of 60 by aerial spraying of crops. Her health problems and immunologic findings are presented in Table IV.

DISCUSSION

This study was designed as an initial evaluation of the peripheral immune system in humans with multiple symptoms following chronic exposure to chlordane/heptachlor in their homes. During its implementation the three subjects exposed to dursban and malathion were added. Four significant observations are reported. First, the patients had an initial flu-like illness followed by persisting multiple organ symptoms. Second, a significant increase in activated T cells (CD26) was present in the peripheral blood. Third, autoantibodies directed against at least five organ systems were detected. Lastly, antimyelin autoantibodies were observed in 9 of 11 subjects given the test

Multiple organ symptomology associated with chlordane/heptachlor exposure has been previously reported.^16,21-23 For the most part these symptoms have been in individuals with various blood dyscrasias, such as aplastic anemia, leukemia, thrombocytopenic purpura, hemolytic anemia, and pernicious anemia. One case report involved an adult male with peripheral neuropathy and evidence of multiple sclerosis.²⁴ Most recently, the incidence of anemia, migraine, neuralgia/neuritis, ovarian and uterine disease, bronchitis, sinusitis, dermatitis, and skin neoplasms were demonstrated to be significantly elevated in humans residing in chlordane treated homes.²⁵ Moreover, in a survey of 6,800 case histories of chemical hypersensitive people, 95 % had an initial flu-like illness, while 48 identified pesticides, including chlordane, as the causative agent in their illness.²⁶ Thus, the initial flu-like illness followed by chronic multiple organ symptoms in these patients is associated with their chlordane/heptachlor exposure. Also, concomitant to these symptoms is an elevation of CD26 cells and the presence of autoantibodies.

The immunologic profiles presented in Tables I and II are consistent with immune activation. The CD26 (Ta1 antigen memory) cells are significantly elevated in the pesticide group. In addition, T cells, although not statistically significant, are elevated in the chlordane patients. The CD26 cells respond to recall antigens and are considered antigen reactive cells.²⁷ Moreover, CD26 and other surface markers are elevated in various autoimmune disorders, such as SLE,

Table IV. This table summarizes the immunologic observations along with initial and chronic symptoms of the 3 patients exposed to organophosphates.

Autoantibodies: Patient 1 (smooth muscle, thyroid microsomal); Patients 2 & 3 (parietal cells, brush boarder),

multiple sclerosis and rheumatoid arthritis.^38-30 Recently, we demonstrated the elevation of CD26 cells in individuals with chronic multi-organ symptoms following exposure to formaldehyde,^1,18,31 isocyanates,³² and TCE contaminated ground water (work in progress). Thus, elevated CD26 cells in these patients are probably casually related to their exposure, multiple symptoms, and the presence of autantibodies.

Environmental chemicals and therapueutic drugs are associated with autoimmune disorders, such as SLE, autoimmune hemolytic anemia, thrombocytopenia, myasthenia gravis, pemphigus, thyroiditis and hepatitis.^33,34 Commensurate with these conditions are multiple organ symptoms, including flu-like conditions, malaise, and symptoms involving the G.I. tract, CNS, skeletal muscle system and skin.^17,18 Thus, the initial flu-like illness, chronic fatigue, malaise, and multi-organ symptoms as well as the high incidence of autoantibodies (Table III) in these patients are highly suggestive of immune pathology and probably a chlordane/heptachlor-induced autoimmune disorder. The mechanism behind the autoimmunity is not known, but may be caused by epoxide metabolites of chlordane/hepatchor.^7-10,35

Neuralgia/neuritis, peripheral neuropathy, and multiple sclerosis findings have been reported with chlordane/heptachlor exposure.^16,24.25 Two of the patients in this study were diagnosed with G-B syndrome. Antimyelin autoantibodies are associated with G-B syndrome, ³⁶ and other demyelinating diseases³⁷ as well as in neuropsychiatric systemic lupus^.38 Moreover, 88 % of adults and 38 % of children have low level (less than 1:4) antimyelin autoantibodies.⁶ In this study, the controls had nondetectable antimyelin autoantibodies at a dilution of 1:8, while 9 of 11 patients were positive. Thus, the presence of antimyelin autoantibodies in these patients suggests that chlordane/heptachlor exposure has caused injury to the myelin sheath. The injury is probably caused by the fat solubility of this pesticide and its metabolites and, as such, may be responsible for the peripheral neuropathy observed in these patients.

In conclusion, individuals chronically exposed to chlordane/heptachlor following application in their homes, develop multiple organ symptoms. These follow an initial flu-like illness. In addition, elevated CD26 cells suggest that an immune activation has occurred in these patients. Commensurate with this elevation is the presence of multi-organ autoantibodies, including ANA and antimyelin. Thus, it seems probable that chronic exposure to chlordane/heptachlor is associated with an autoimmune disorder that heretofore has been described. Perhaps, autoimmunity is at the basis of their chronic health problems.

The patients in this study, with the exception of the pheocytochroma and the two G-B syndromes, have had negative diagnostics with respect to their chronic multiple symptomology. For the most part their attending physicians were either puzzled by the illness or denied that chordane/heptachlor could cause their health problems. Psychiatric evaluation in some cases was recommended. Perhaps, their multiple health problems do have an underlying etiology, such as immune activation and autommunity. For example, multiple organ symptoms do occur in systemic LE and mixed connective tissue disease.¹⁷

Appendix:

When this paper was published we had no information on the concentrations of chlordane in the homes of the exposed. In this appendix we present information on Chlordane levels in two of the home totaling eight people, 4 adults and 4 of their children.

Family One: Consisted of two adults and two children. One of the children (male) was diagnosed with malignant pheocytochroma. The following tests were performed on this family: Bulk, wipe and clothing tested for chlordane and aldrin by Dr. Robert Simon Washington, Analytical Laboratory, Chantilly, VA), and adipose and serum concentrations of heptachlor epoxide, oxychlordane and transnonachlor. The new construction home was broadcast sprayed with Gold Crest Termiticide on 7//2/7/79 with the family taking occupancy in late September, 1979. The mother had the following pregnancy histories: daughter born 1/8/80; Mid-September, 1980 aborted a deformed fetus; 1/13/82 gave birth to son (diagnosed later with pheocytochroma). Results of Bulk Samples in home showed technical chlordane (1.2, 1.6, 0.2 ppm, heptachlor (0.3 ppm) and aldlrin (1.0, 0.3, 0.04 ppm). Wipe samples: Technical chlordane (n = 8; mean of 33.6 ug/ft2); heptachlor (n = 4; mean of 1.0 ug/ft2); aldrin (n = 13, mean of 11.6 ug/2). Father’s shirts (n = 2) had the following: Heptachlor (mean of 1.5 ug/ft2); chlordane (mean of 2.4 ug/ft2); aldrin (not detected); Father’s pant cuff: heptachlor (0.4 ug/ft2), chlordane (25.6 ug/ft2); aldrin (not detected); daughter’s underwear: heptachlor (0.7 ug/ft2); chlordane (9.6 ug/ft2); aldrin (3.2 ug/ft2); Son’s underwear: heptachlor (1.7 ug/ft2); chlordane (12.6 ug/ft2); aldrin (not detectes. Serum concentrations for heptachlor epoxide, oxychlordane and transnonachlor were not detected. Adipose concentrations of the three metabolites for the father, mother, daughter and son were: 30, 67, 101 ppb; 41, 69, 48 ppb; 27, 58, 79 ppb; and 33, 73, 114 ppb, respectively. The transnonachlor was at elevated levels (above the average level found in the general population) for the father, son and daughter.

Family Two: This constituted a family of four, for which no additional health history is obtainable at the writing of this appendix. The home was a raised foundation. The home was tested by the Texas Department of Health on 10/3/84. The family had moved into the home approximately 18 months prior to air sampling. The house had prior treatment for termiticide infestation. Air sampling was accomplished with MSA Mode G personal sampling pumps with SKC Chromosorb sample tubes. The samples were analyzed for chlordane and heptachlor by high performance liquid chromatography. The tests results showed chlordane at 185 ug/m3 (den) and 92 ug/m3 (teenager’s room). The Texas Department of Health report stated the following: Your son being treated at Childrens Medical Center in Dallas has experienced headaches, weight loss, irritability, back pain, and stomach aches, a younger son experiences severe headaches, mild nervous system depression, and extreme hyperactivity.

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