Jack D. Thrasher

Immunological abnormalities in humans chronically exposed to chlorpyrifos

Abstract Twenty-nine individuals with chronic health complaints following exposure to chlorpyrifos were compared with 3 control groups (i.e., 1 positive and 2 negative) with respect to the following: (1) peripheral lymphocyte phenotypes; (2) autoantibodies (nucleic acids and nucleoproteins, parietal cell, brush border, mitochondria, smooth muscle, thyroid gland, and central nervous system/peripheral nervous system myelin); (3) mitogenesis to phytohemagglutinin and concanavillin. The data revealed an increase in CD26 expression, a decrease in percentage of CD5 phenotype, decreased mitogenesis in response to phytohemagglutinin and concanavillin, and an increased frequency of autoantibodies. The alterations in these peripheral blood markers were unaffected by medications, age, sex, or season. The authors concluded that chronic exposure to chlorpyrifos causes immunological changes.

Embryo Toxicity and Teratogenicity of Formaldehyde

Abstract C-14 formaldehyde crosses the placenta and enters fetal tissues. The incorporated radioactivity is higher in fetal organs (i.e., brain and liver) than in maternal tissues. The incorporation mechanism has not been studied fully, but formaldehyde enters the single-carbon cycle and is incorporated as a methyl group into nucleic acids and proteins. Also, formaldehyde reacts chemically with organic compounds (e.g., deoxyribonucleic acid, nucleosides, nucleotides, proteins, amino acids) by addition and condensation reactions, thus forming adducts and deoxyribonucleic acid-protein crosslinks. The following questions must be addressed: What adducts (e.g., N-methyl amino acids) are formed in the blood following formaldehyde inhalation? What role do N-methyl-amino adducts play in alkylation of nuclear and mitochondrial deoxyribonucleic acid, as well as mitochondrial peroxidation? The fact that the free formaldehyde pool in blood is not affected following exposure to the chemical does not mean that formaldehyde is not involved in altering cell and deoxyribonucleic acid characteristics beyond the nasal cavity. The teratogenic effect of formaldehyde in the English literature has been sought, beginning on the 6th day of pregnancy (i.e., rodents) (Saillenfait AM, et al. Food Chem Toxicol 1989, pp 545–48; Martin WJ. Reprod Toxicol 1990, pp 237–39; Ulsamer AC, et al. Hazard Assessment of Chemicals; Academic Press, 1984, pp 337–400; and U.S. Department of Health and Human Services. Toxicological Profile of Formaldehyde; ATSDR, 1999 [references 1–4, respectively, herein]). The exposure regimen is critical and may account for the differences in outcomes. Pregnant rats were exposed (a) prior to mating, (b) during mating, (c) or during the entire gestation period. These regimens (a) increased embryo mortality; (b) increased fetal anomalies (i.e., cryptochordism and aberrant ossification centers); (c) decreased concentrations of ascorbic acid; and (d) caused abnormalities in enzymes of mitochondria, lysosomes, and the endoplasmic reticulum. The alterations in enzymatic activity persisted 4 mo following birth. In addition, formaldehyde caused metabolic acidosis, which was augmented by iron deficiency. Furthermore, newborns exposed to formaldehyde in utero had abnormal performances in open-field tests. Disparities in teratogenic effects of toxic chemicals are not unusual. For example, chlorpyrifos has not produced teratogenic effects in rats when mothers are exposed on days 6–15 (Katakura Y, et al. Br J Ind Med 1993, pp 176–82 [reference 5 herein]) of gestation (Breslin WJ, et al. Fund Appl Toxicol 1996, pp 119–30; and Hartley TR, et al. Toxicol Sci 2000, pp 100–08 [references 6 and 7, respectively, herein]). However, either changing the endpoints for measurement or exposing neonates during periods of neurogenesis (days 1–14 following birth) and during subsequent developmental periods produced adverse effects. These effects included neuroapoptosis, decreased deoxyribonucleic acid and ribonucleic acid synthesis, abnormalities in adenylyl cyclase cascade, and neurobehavioral effects (Johnson DE, et al. Brain Res Bull 1998, pp 143–47; Lassiter TL, et al. Toxicol Sci 1999, pp 92–100; Chakraborti TK, et al. Pharmacol Biochem Behav 1993, pp 219–24; Whitney KD, et al. Toxicol Appl Pharm 1995, pp 53–62; Chanda SM, et al. Pharmacol Biochem Behav 1996, pp 771–76; Dam K, et al. Devel Brain Res 1998, pp 39–45; Campbell CG, et al. Brain Res Bull 1997, pp 179–89; and Xong X, et al. Toxicol Appl Pharm 1997, pp 158–74 [references 8–15, respectively, herein]). Furthermore, the terata caused by thalidomide is a graphic human example in which the animal model and timing of exposure were key factors (Parman T, et al. Natl Med 1999, pp 582–85; and Brenner CA, et al. Mol Human Repro 1998, pp 887–92 [references 16 and 17, respectively, herein]). Thus, it appears that more sensitive endpoints (e.g., enzyme activity, generation of reactive oxygen species, timing of exposure) for the measurement of toxic effects of environmental agents on embryos, fetuses, and neonates are more coherent than are gross terata observations. The perinatal period from the end of organogenesis to the end of the neonatal period in humans approximates the 28th day of gestation to 4 wk postpartum. Therefore, researchers must investigate similar stages of development (e.g., neurogenesis occurs in the 3rd trimester in humans and neonatal days occur during days 1–14 in rats and mice, whereas guinea pigs behave more like humans). Finally, screening for teratogenic events should also include exposure of females before mating or shortly following mating. Such a regimen is fruitful inasmuch as environmental agents cause adverse effects on ovarian elements (e.g., thecal cells and ova [nuclear-deoxyribonucleic acid and mitochondrial deoxyribonucleic acid]), as well as on zygotes and embryos before implantation. Mitochondrial deoxyribonucleic acid mutations and deletions occur in human oocytes and embryos (Parman T, et al. Natl Med 1999, pp 582–85; and Brenner CA, et al. Mol Human Repro 1998, pp 887–92 [references 16 and 17, respectively, herein]). Thus, it is likely that xenobiotics directly affect n-deoxyribonucleic acid and/or mitochondrial deoxyribonucleic acid in either the ovum or the zygote/embryo or both (Thrasher JD. Arch Environ Health 2000, pp 292–94 [reference 18 herein]), and they could account for the increasing appearance of a variety of mitochondrial diseases, including autism (Lomard L. Med Hypotheses 1998, pp 497–99; Wallace EC. Proc Natl Acad Sci 1994, pp 8730–46; and Giles RE, et al. Proc Natl Acad Sci 1980, pp 6715–19 [references 19–21, respectively, herein]). Two cases of human birth defects were reported in formaldehyde-contaminated homes (Woodbury MA, et al. Formaldehyde Toxicity 1983; pp 203–11 [reference 22 herein]). One case was anencephalic at 2.76 ppm, and the other defect at 0.54 ppm was not characterized. Further observations on human birth defects are recommended.

Immune Activation and Autoantibodies in Humans with Long-Term Inhalation Exposure to Formaldehyde

Four groups of patients with long-term inhalation exposure to formaldehyde (HCHO) were compared with controls who had short-term periodic exposure to HCHO. The following were determined for all groups: total white cell, lymphocyte, and T cell counts; T helper/suppressor ratios; total Ta1+, IL2+, and B cell counts; antibodies to formaldehyde-human serum albumin (HCHO-HSA) conjugate and autoantibodies. When compared with the controls, the patients had significantly higher antibody titers to HCHO-HSA. In addition, significant increases in Ta1+, IL2+, and B cells and autoantibodies were observed. Immune activation, autoantibodies, and anti-HCHO-HSA antibodies are associated with long-term formaldehyde inhalation.

The biocontaminants and complexity of damp indoor spaces: more than what meets the eyes

Nine types of biocontaminants in damp indoor environments from microbial growth are discussed: (1) indicator molds; (2) Gram negative and positive bacteria; (3) microbial particulates; (4) mycotoxins; (5) volatile organic compounds, both microbial (MVOCs) and non-microbial (VOCs); (6) proteins; (7) galactomannans; (8) 1-3-β-D-glucans (glucans) and (9) lipopolysaccharides (LPS — endotoxins). When mold species exceed those outdoors contamination is deduced. Gram negative bacterial endotoxins, LPS in indoor environments, synergize with mycotoxins. The gram positive Bacillus species, Actinomycetes (Streptomyces, Nocardia and Mycobacterium), produce exotoxins. The Actinomycetes are associated with hypersensitivity pneumonitis, lung and invasive infections. Mycobacterial mycobacterium infections not from M. tuberculosis are increasing in immunocompetent individuals. In animal models, LPS enhance the toxicity of roridin A, satratoxins G and aflatoxin B1 to damage the olfactory epithelium, tract and bulbs (roridin A, satratoxin G) and liver (aflatoxin B1). Aflatoxin B1 and probably trichothecenes are transported along the olfactory tract to the temporal lobe. Co-cultured Streptomyces californicus and Stachybotrys chartarum produce a cytotoxin similar to doxorubicin and actinomycin D (chemotherapeutic agents). Trichothecenes, aflatoxins, gliotoxin and other mycotoxins are found in dust, bulk samples, air and ventilation systems of infested buildings. Macrocyclic trichothecenes are present in airborne particles <2 μm. Trichothecenes and stachylysin are present in the sera of individuals exposed to S. chartarum in contaminated indoor environments. Haemolysins are produced by S. chartarum, Memnoniella echinata and several species of Aspergillus and Penicillium. Galactomannans, glucans and LPS are upper and lower respiratory tract irritants. Gliotoxin, an immunosuppressive mycotoxin, was identified in the lung secretions and sera of cancer patients with aspergillosis produced by A. fumigatus, A. terreus, A. niger and A. flavus.

Mixed mold mycotoxicosis: immunological changes in humans following exposure in water-damaged buildings.

The study described was part of a larger multicenter investigation of patients with multiple health complaints attributable to confirmed exposure to mixed-molds infestation in water-damaged buildings. The authors present data on symptoms; clinical chemistries; abnormalities in pulmonary function; alterations in T, B, and natural killer (NK) cells; the presence of autoantibodies (i.e., antinuclear autoantibodies [ANA], autoantibodies against smooth muscle [ASM], and autoantibodies against central nervous system [CNS] and peripheral nervous system [PNS] myelins). A total of 209 adults, 42.7 ± 16 yr of age (mean ± standard deviation), were examined and tested with (a) self-administered weighted health history and symptom questionnaires; (b) standardized physical examinations; (c) complete blood counts and blood and urine chemistries; (d) urine and fecal cultures; (e) thyroid function tests (T4, free T3); (f) pulmonary function tests (forced vital capacity [FVC], forced expiratory volume in 1 sec [FEV1.0], and forced expiratory flow at 25%, 50%, 75%, and 25–75% of FVC [FEF25, FEF50, FEF75, and FEF25–75]); (g) peripheral lymphocyte phenotypes (T, B, and NK cells) and mitogenesis determinations; and (h) a 13-item autoimmune panel. The molds-exposed patients reported a greater frequency and intensity of symptoms, particularly neurological and inflammatory symptoms, when compared with controls. The percentages of exposed individuals with increased lymphocyte phenotypes were: B cells (CD20+), 75.6%; CD5+CD25+, 68.9%; CD3+CD26+, 91.2%; CD8+HLR–DR+, 62%; and CD8+CD38+, 56.6%; whereas other phenotypes were decreased: CD8+CD11b+, 15.6% and CD3–CD16+CD56+, 38.5%. Mitogenesis to phytohemagglutinin was decreased in 26.2% of the exposed patients, but only 5.9 % had decreased response to concanavalin A. Abnormally high levels of ANA, ASM, and CNS myelin (immunoglobulins [Ig]G, IgM, IgA) and PNS myelin (IgG, IgM, IgA) were found; odds ratios for each were significant at 95% confidence intervals, showing an increased risk for autoimmunity. The authors conclude that exposure to mixed molds and their associated mycotoxins in water-damaged buildings leads to multiple health problems involving the CNS and the immune system, in addition to pulmonary effects and allergies. Mold exposure also initiates inflammatory processes. The authors propose the term “mixed mold mycotoxicosis” for the multisystem illness observed in these patients.

Low-level hydrogen sulfide and central nervous system dysfunction

Forty-nine adults living in Lovington, Tatum, and Artesia, the sour gas/oil sector of Southeastern New Mexico, were tested for neurobehavioral impairment. Contributing hydrogen sulfide were (1) an anaerobic sewage plant; (2) two oil refineries; (3) natural gas/oil wells and (4) a cheese-manufacturing plant and its waste lagoons. Comparisons were to unexposed Wickenburg, Arizona, adults. Neurobehavioral functions were measured in 26 Lovington adults including 23 people from Tatum and Artesia, New Mexico, and 42 unexposed Arizona people. Participants completed questionnaires including chemical exposures, symptom frequencies and the Profile of Mood States. Measurements included balance, reaction time, color discrimination, blink reflex, visual fields, grip strength, hearing, vibration, problem solving, verbal recall, long-term memory, peg placement, trail making and fingertip number writing errors (FTNWE). Average numbers of abnormalities and test scores were adjusted for age, gender, educational level, height and weight, expressed as percent predicted (% pred) and compared by analysis of variance (ANOVA). Ages and educational attainment of the three groups were not statistically significantly different (ssd). Mean values of Lovington residents were ssd from the unexposed Arizona people for simple and choice reaction times, balance with eyes open and closed, visual field score, hearing and grip strength. Culture Fair, digit symbol substitution, vocabulary, verbal recall, peg placement, trail making A and B, FTNWE, information, picture completion and similarities were also ssd. The Lovington adults who averaged 11.8 abnormalities were ssd from, Tatum—Artesia adults who had 3.6 and from unexposed subjects with 2.0. Multiple source community hydrogen sulfide exposures impaired neurobehavioral functions.

Mold and mycotoxins: effects on the neurological and immune systems in humans.

Publisher Summary There can be a complexity of health problems associated with multiple mold exposure. This chapter describes the most recent neuroimmune mechanisms of diseases caused by molds and mycotoxins in humans. The exact biological and chemical actions through which these mechanisms unfold are not completely understood. However, molds do produce metabolites such as mycotoxins and shed antigenic materials—namely, spores, hyphae, extracellular polysaccharides, and enzymes—that are toxic and/or cause immunologic responses. The chapter discusses detailed health and environmental history, environmental monitoring data, physical examinations, routine clinical chemistries, measurements of lymphocyte phenotypic markers, antibodies to molds, mycotoxins, neuronal antigen antibodies, leukocyte apoptosis, nerve conduction studies (NCS), brainstem auditory evoked potentials (BAER), visual evoked responses (VER), and other neurological testing. The illness of these individuals is referred to as a “mold mycotoxicosis,” and it involves the immune system, the lungs, the central and peripheral nervous systems, and generalized inflammatory and irritant responses to exposure to spores, hyphal fragments, mycotoxins, solvents, and other byproducts.

A Water-Damaged Home and Health of Occupants: A Case Study

A family of five and pet dog who rented a water-damaged home and developed multiple health problems. The home was analyzed for species of mold and bacteria. The diagnostics included MRI for chronic sinusitis with ENT and sinus surgery, and neurological testing for neurocognitive deficits. Bulk samples from the home, tissue from the sinuses, urine, nasal secretions, placenta, umbilical cord, and breast milk were tested for the presence of trichothecenes, aflatoxins, and Ochratoxin A. The family had the following diagnosed conditions: chronic sinusitis, neurological deficits, coughing with wheeze, nose bleeds, and fatigue among other symptoms. An infant was born with a total body flare, developed multiple Cafe-au-Lait pigmented skin spots and diagnoses with NF1 at age 2. The mycotoxins were detected in bulk samples, urine and nasal secretions, breast milk, placenta, and umbilical cord. Pseudomonas aueroginosa, Acinetobacter, Penicillium, and Aspergillus fumigatus were cultured from nasal secretions (father and daughter). RT-PCR revealed A. fumigatus DNA in sinus tissues of the daughter. The dog had 72 skin lesions (sebaceous glands and lipomas) from which trichothecenes and ochratoxin A. were detected. The health of the family is discussed in relation to the most recent published literature regarding microbial contamination and toxic by-products present in water-damaged buildings.

Antibodies to molds and satratoxin in individuals exposed in water-damaged buildings.

Immunoglobulin (Ig)A, IgM, and IgG antibodies against Penicillium notatum, Aspergillus niger, Stachybotrys chartarum, and satratoxin H were determined in the blood of 500 healthy blood donor controls, 500 random patients, and 500 patients with known exposure to molds. The patients were referred to the immunological testing laboratory for health reasons other than mold exposure, or for measurement of mold antibody levels. Levels of IgA, IgM, and IgG antibodies against molds were significantly greater in the patients (p < 0.001 for all measurements) than in the controls. However, in mold-exposed patients, levels of these antibodies against satratoxin differed significantly for IgG only (p < 0.001), but not for IgM or IgA. These differences in the levels of mold antibodies among the 3 groups were confirmed by calculation of z score and by Scheffés significant difference tests. A general linear model was applied in the majority of cases, and 3 different subsets were formed, meaning that the healthy control groups were different from the random patients and from the mold-exposed patients. These findings indicated that mold exposure was more common in patients who were referred for immunological evaluation than it was in healthy blood donors. The detection of antibodies to molds and satratoxin H likely resulted from antigenic stimulation of the immune system and the reaction of serum with specially prepared mold antigens. These antigens, which had high protein content, were developed in this laboratory and used in the enzyme-linked immunosorbent assay (ELISA) procedure. The authors concluded that the antibodies studied are specific to mold antigens and mycotoxins, and therefore could be useful in epidemiological and other studies of humans exposed to molds and mycotoxins.

Neural autoantibodies and neurophysiologic abnormalities in patients exposed to molds in water-damaged buildings

Adverse health effects of fungal bioaerosols on occupants of water-damaged homes and other buildings have been reported. Recently, it has been suggested that mold exposure causes neurological injury. The authors investigated neurological antibodies and neu-rophysiological abnormalities in patients exposed to molds at home who developed symptoms of peripheral neuropathy (i.e., numbness, tingling, tremors, and muscle weakness in the extremities). Serum samples were collected and analyzed with the enzyme-linked im-munosorbent assay (ELISA) technique for antibodies to myelin basic protein, myelin-associ-ated glycoprotein, ganglioside GM 1, sulfatide, myelin oligodendrocyte glycoprotein, α-B-crystallin, chondroitin sulfate, tubulin, and neurofilament. Antibodies to molds and mycotoxins were also determined with ELISA, as reported previously. Neurophysiologic evaluations for latency, amplitude, and velocity were performed on 4 motor nerves (median, ulnar, peroneal, and tibial), and for latency and amplitude on 3 sensory nerves (median, ulnar, and sural). Patients with documented, measured exposure to molds had elevated titers of antibodies (immunoglobulin [Ig]A, IgM, and IgG) to neural-specific antigens. Nerve conduction studies revealed 4 patient groupings: (1) mixed sensory-motor polyneuropathy (n = 55, abnormal), (2) motor neuropathy (n = 17, abnormal), (3) sensory neuropathy (n = 27, abnormal), and (4) those with symptoms but no neurophysiological abnormalities (n = 20, normal controls). All groups showed significantly increased autoantibody titers for all iso-types (IgA, IgM, and IgG) of antibodies to neural antigens when compared with 500 healthy controls. Groups 1 through 3 also exhibited abnormal neurophysiologic findings. The authors concluded that exposure to molds in water-damaged buildings increased the risk for development of neural autoantibodies, peripheral neuropathy, and neurophysiologic abnormalities in exposed individuals.