CHRONIC EXPOSURE to low concentrations of formaldehyde (HCHO) in indoor air is
common in occupational and nonoccupational environments.^1-3 These individuals have many health complaints that involve the skin, eyes, upper and lower respiratory tract, central nervous system, and the gastro-intestinal tract.^1-6 Symptoms suggest systemic involvement and may reflect that HCHO acts as a hapten to produce antibodies.^7-12

Individuals chronically exposed to low concentrations of indoor HCHO have fatigue, headache, vertigo, and upper and lower respiratory irritation that are difficult to classify by a single mechanism.^2,7 Early observations, however, indicate that HCHO is a hapten, binding to human serum albumin (HSA) to form a new antigenic determinant, F-HSA.⁸ Recently, IgE antibodies to F-HSA have been demonstrated in anaphylactic shock caused by HCHO in long-term hemodialysis.⁹ Also, IgG and IgM hemolytic anti-HCHO red blood cell (RBC) antibodies are present under similar hemodialysis conditions.^10,11 Moreover, an anti-F-HSA IgG has been produced in dogs by the intravenous administration of HCHO and F-DSA.¹² More recently, IgG antibodies to F-HSA have been found in asymptomatic renal dialysis patients, IgE antibodies in two nurses with a history of HCHO asthma, and no antibodies in two physicians with HCHO rhinitis.¹³ Airborne HCHO apparently conjugates with proteins of the upper respiratory tract.¹⁴ This study examined for antibodies to F-HAS in subjects chronically exposed to low concentrations of HCHO in indoor air. In addition, the numbers and blastogenesis of peripheral T and B lymphocytes were studied.

Materials and Methods

HCHO-exposed subjects. Seven of the subjects resided in mobile homes. One worked in the laboratory and lived in a newly decorated energy-efficient apartment. They had multiple health complaints^1-6 recorded on a symptom checklist in their home developed by Godish.¹⁵ A medical history and physical examination showed that three subjects had a history of allergies, but otherwise no obvious explanation for their health complaints. Physical examinations and diagnostic tests (complete blood count, urinalysis, SMAC 24) were normal. One male who was 63 yr of age smoked 20 cigarettes per day, while one female, age 28, smoked 0-5 cigarettes per day. The subjects had lived 2-7 yr in their current residences. The exposed subjects included five females who were 28, 55, 58, 65 and 75 yr of age, and three males who were 63, 70 and 70 yr of age. They comprised three married couples and two single females.

Unexposed (control) subjects. The control subjects consisted of four laboratory personnel and four elderly patients. The laboratory personnel included three men, ages 42, 47 and 35 yr, and a 35 yr-old woman, all of whom were asymptomatic. One (age 47) smoked two packs of cigarettes per day. The four elderly patients (i.e. two couples) were treated for whiplash (male 75); probable stroke (male 75); migraine headaches and multiple pollen and grass allergies (female, 63); and Parkinson's disease (female, 67).

HCHO measurements. HCHO was measured by the County Health Departments in Fresno (two mobile homes) and Los Angeles (two mobile homes) counties, utilizing the Na bisulfite impingement method recommended by the Occupational Safety and Health Administration (OSHA). Three of the homes had two separate measurements, while the third had one HCHO measurement. The concentrations of HCHO in ppm and yr of occupancy were, respectively: 0.07, 0.16 (6 yr); 0.110, 0.117 (6 yr); 0.33, 0.33 (7 yr); and 0.55 (yr). Formaldehyde measurements were not done in the laboratory worker's apartment nor in the homes of the control subjects, which were at least 20 yr old. It was assumed that HCHO concentrations would be similar to those reported and estimated by the Environmental Protection Agency,¹⁶ i.e, a mean value of 0.03 ppm.

HCHO antibodies. Formaldehyde (F) was conjugated to chromatographically pure human serum albumin (Sigma) following the procedures of Patterson et al.^12,13 Lymphocytes were separated form venous blood of exposed and control subjects and immediately used for T and B cell enumeration and blastogenesis. The sera were used to measure IgG and IgE antibodies to F-HSA by the methods of Patterson et al.^12,13

The anti-F-HSA antibodies were determined by the ELISAprocedure.¹⁷ In brief, micro ELISA plates (Dynatech Laboratories, Alexandria, VA) were coated with various dilutions of either F-HSA or HSA as antigen sources. After incubation for 2 hr, the plates were washed with PBS-Tween (0.5%, Sigma). Dilutions of test and control sera were made in PBS-Tween and 200 ul alkaline phosphatase labeled anti-human IgG anti-human IgE were added. The plates were then incubated for 12 hr at 4 ^oC, washed in PBS-Tween, and 200 ul of substrate (paranitrophenyl phosphate) was added. Thirty minutes later the reaction was stopped by adding 50 ul of 3 mol/L of sodium hydroxide. The optical densities were read at 405 nm on an automatic ELISA reader (Dynatech, Alexandria, VA). ELISA end point titers were read when the optical density reading of the test sera (HCHO exposed) was 2 times that of the negative control (HSA) wells.

T and B enumeration and blastogenesis. T and B cells were enumerated by E- and EAC-rosetting method, respectively.18 In both methods, lymphocytes with binding of more that two sheep RBCs were considered positive. The blastogenesis of T and B cells was measured by mitogen activation using phytohemagglutin (T cells) and pokeweed mitogen (T and B cells). The lymphocytes from both exposed and control subjects were culture at 37 ^oC in an atmosphere of 95 % air and 5 % carbon dioxide. The cultures were done in quadruplicate in wells of flat bottom micro-titer plates with optimum concentrations of either PHA (4.0 ug/ml) or PWM (8.0 ug/ml). After 48 hr the cells were labeled with 2 uCi of tritiated thymidine per well for 24 hr. The lymphocytes were harvested and the incorporation of the labeled thymidine into cellular DNA was measured by liquid scintillation counting. The data were expressed as cpm + standard deviation for each of the exposed and nonexposed subjects. The variability in the blastogenesis assay was less than 20 %.

Results

F-HSA antibodies. IgE antibodies to F-HSA were not detected in exposed and unexposed subjects (Table 1). IgG antibodies to F-HSA ranged from 1:8 to 1:256 in the mobile home occupants. Anti-F-HSA antibodies were not detected in seven of the control subjects (1:4) and were 1:8 in the subject with Parkinson's disease. No antibodies (IgE and IgG) were detected in the micro ELISA wells coated with HSA. Thus, cross reactivity in the assay for IgG antibodies was absent.

B lymphocytes in the HCHO-exposed individuals ranged from 11-15%, mean 12.6+1.6%. Controls ranged from 12-18%, mean 14.75+2.1%. The difference between the means was not significant, p >.02<.05.

T and B cell blastogenesis. T cell blastogenesis, as determined by PHA stimulation, was lower in the exposed group than in the controls (Table 2). The uptake of 3H-Tdr in the HCHO-exposed subjects averaged 17,882+2,293 cpm, while that for the controls was 28,576+3,807 cpm. The difference between the means was significant, p >.001<.01.

T and B cell blastogenesis, as determine PWM stimulation, appeared lower in the experimental group vs. the controls (Table 2). The uptake of 3H-Tdr averaged 9,698+1,441 cpm in the HCHO-exposed individuals, and controls were 11,279+1,711 cpm. The difference between the means was significant, p >05 <.01.

Discussion

Inhaled HCHO is incorporated into nucleic acids or is covalently bound to proteins in the nasal mucosa of rats.¹⁴ Inhalation exposure causes nasal epithelial cell proliferation and metaplasia, congestion, and secretion in rodents and monkeys.^20-22 Human subjects experience rhinitis, congestion, and epistaxis following chronic HCHO exposure in their homes.^2,4-6 Thus, damage to mucosal tissue, including capillary beds, could facilitate HCHO conjugation with blood proteins. IgG antibodies found in this study appear to be directed towards F-HSA conjugates. This is deduced because antigen/antibody cross reactivity to HSA was not observed and IgE and IgG antibodies F-HSA were absent or minimal in the asymptomatic controls. Thus, anti F-HSA antibodies probably indicate exposure to HCHO. IgG subtype 4 antibodies are under investigation in individuals exposed to airborne HCHO.

IgE antibodies may have escaped detection because they are present in trace amounts and have a half-life of approximately 2 days.²³ At the time of this investigation these facts were not considered. Subsequently, IgE anti-F-HSA antibodies have been detected.²⁴

Modulation of cell-mediated immunity (CMI) by HCHO has been demonstrated in rodents. Mice exposed for 21 days (6 hr/day), 15 ppm) showed enhanced macrophage H₂O₂ production. In addition, resistance to I. monocytogenes challenge was significantly increased.²⁵ Conversely, F-344 rats in a 4-wk study (HCHO for 6 hr/day at 3, 16, 32 ppm) had depressed numbers of peripheral B cells at the higher concentrations and decreased production of antibodies to sheep RBCs.²⁶ In this study, the exposed subjects had a decreased percentage of T cells when compared to the controls. Similar observations have been reported in humans exposed to a variety of environmental agents.²⁷ Although mechanisms responsible for changes in the CMI are poorly understood, they may be related to chronic antigenic stimulation by F-HSA.^24,28 Also, PWM stimulates both B and T cells, while PHA induces mitosis in T cells. Thus, the difference between the response of T and B cells of these subjects is interpreted as T cell rather than B cell modulation of CMI. In addition, B cell modulation may be minimal in humans.^24,28

Submitted for publication June 15, 1987, revised, accepted for publication July 2, 1987. Requests for reprints should be sent to Jack D. Thrasher, Ph.D., Thrasher & Associates 11330 Quail Creek Rd., Northridge, CA 91326

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