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7—
Immunologic Effects

This chapter discusses the immunologic effects attributed to persistent organochlorines and other hormonally active agents (HAAs). Effects of specific HAAs (such as the halogenated aromatic hydrocarbons (HAHs), dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), endrin, aldrin, dieldrin, lindane, chlordane, toxaphene, endosulfan, and hexachlorobenzene) observed in laboratory studies and to a lesser extent in field and human studies are compared and correlated with information on exposure. Because these agents are postulated to act by means of hormonally mediated mechanisms, a brief discussion of how hormones affect the immune system is presented below. As more information becomes available, this will provide a context for evaluating the immunologic effects of HAAs.

It has been well documented that immunity can be modulated by hormones (Grossman 1984, 1985). The presence of steroid hormone receptors in a strikingly wide variety of immunologic tissues is a strong indication that cells and tissues of the immune system must be targets for steroid hormones, and that steroid hormones elicit regulatory effects in these cells and tissues. The heterogeneity of responses caused by interactions of various steroid hormones (such as corticoids, estrogens, androgens, and progestins) between the immune and endocrine systems has been documented (Grossman 1984, 1985, 1989, 1994: Grossman et al. 1991; Berczi 1994; Chapman and Michael 1994; Dardeene and Savino 1994; Fabris 1994; McCruden and Stimson 1994; Rivier 1994; Wira et al. 1994). Thus, a variety of stimuli (including exposure to environmental toxicants) could mediate nonspecific, stress like effects.

In the case of steroid hormones, it is clear that the actions of estrogens and androgens are important in the reported differences in immune response between male and female laboratory animals (Batchelor and Chapman 1965: Terres et al.continue



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Page 186 7— Immunologic Effects This chapter discusses the immunologic effects attributed to persistent organochlorines and other hormonally active agents (HAAs). Effects of specific HAAs (such as the halogenated aromatic hydrocarbons (HAHs), dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), endrin, aldrin, dieldrin, lindane, chlordane, toxaphene, endosulfan, and hexachlorobenzene) observed in laboratory studies and to a lesser extent in field and human studies are compared and correlated with information on exposure. Because these agents are postulated to act by means of hormonally mediated mechanisms, a brief discussion of how hormones affect the immune system is presented below. As more information becomes available, this will provide a context for evaluating the immunologic effects of HAAs. It has been well documented that immunity can be modulated by hormones (Grossman 1984, 1985). The presence of steroid hormone receptors in a strikingly wide variety of immunologic tissues is a strong indication that cells and tissues of the immune system must be targets for steroid hormones, and that steroid hormones elicit regulatory effects in these cells and tissues. The heterogeneity of responses caused by interactions of various steroid hormones (such as corticoids, estrogens, androgens, and progestins) between the immune and endocrine systems has been documented (Grossman 1984, 1985, 1989, 1994: Grossman et al. 1991; Berczi 1994; Chapman and Michael 1994; Dardeene and Savino 1994; Fabris 1994; McCruden and Stimson 1994; Rivier 1994; Wira et al. 1994). Thus, a variety of stimuli (including exposure to environmental toxicants) could mediate nonspecific, stress like effects. In the case of steroid hormones, it is clear that the actions of estrogens and androgens are important in the reported differences in immune response between male and female laboratory animals (Batchelor and Chapman 1965: Terres et al.continue

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Page 187 1968; Grossman 1984, 1985, 1989, 1994; Grossman et al. 1991). Termed "immunologic sexual dimorphism," the results of the actions are a general increase in humoral immunity in females compared with males (Batchelor and Chapman 1965; Terres et al. 1968) and differences in some cell-mediated immune responses (Graff et al. 1966, 1969; Kittas and Henry 1979, 1980) in females and males. For example, females tend to be far more susceptible than males to such autoimmune diseases as Hashimoto's disease (Tunbridge et al. 1977: Grossman et al. 1991), Grave's disease (Grossman et al. 1991), rheumatoid arthritis (Vandenbroucke 1982; Vandenbroucke et al. 1982; Lotz and Vaughan 1988; Grossman et al. 1991), systemic lupus erythematosus (Roubinian et al. 1979a,b; Lahita 1985; Grossman et al. 1991), thyroid disease (Tunbridge et al. 1977), and demyelinating disease (Arnason and Richman 1969). Immunologic differences are also observed between pregnant and nonpregnant females. Immune responses in pregnant women are depressed compared with nonpregnant women. This depression of the immune system might be necessary during pregnancy to prevent fetal rejection and abortion before term (Grossman and Roselle 1987), and might be due, in part, to the presence of sex steroids that are elevated during pregnancy. However, it also could be partly responsible for the reported increases in the susceptibility of pregnant women to such infectious diseases as smallpox, polio, viral hepatitis, varicella-zoster, influenza, cytomegalovirus, and pulmonary and systemic mycoses (Grossman and Roselle 1987). Diethylstilbestrol (DES) has been studied extensively in humans, and this estrogen has been shown to alter immunity (Dodds et al. 1938; Ablin et al. 1974; Korach et al. 1978; Dean et al. 1980; Kalland and Forsberg 1981; Haukaas et al. 1982; Fugmann et al. 1983; Luster et al. 1984; Morahan et al. 1984; Pung et al. 1984, 1985; Noller et al. 1988). These studies provide examples of how hormones can affect the immune system, but whether all HAAs act in this manner remains to be determined. HAAs and Steroid Hormones HAAs and Lymphatic Tissue Structure Some reports of architectural changes in primary and secondary lymphatic tissues exposed to HAAs are available. Of particular interest are studies that describe thymic atrophy, since the thymus is a major site of early T-lymphocyte development, as well as a source of immunologic regulatory hormones (Dardeene and Savino 1994) in the adult. Thus, it follows that disorganization of thymic structure in the embryo could also result in immunologic abnormalities. In mammalian species, thymic atrophy and disruption of the secondary lymphatic organs has been generally observed as a result of PCB exposure (McKinney et al. 1976: Safe 1985; Thomas and Faith 1985), and these compounds can alter lymphoidcontinue

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Page 188 development of thymus and bursa (Andersson et al. 1991). Notably, TCDD promotes thymic involution in fish (Spitsbergen et al. 1986) and mice (Luster and Rosenthal 1986; Kerkvliet et al. 1990) and cellular depletion in thymus, spleen, and lymph nodes (Clark et al. 1981). The action of some HAAs, such as TCDD and some PCBs, are mediated through the aryl hydrocarbon (Ah) receptor mechanism (Kerkvliet et al. 1990: Andersson et al. 1991; Kerkvliet and Burleson 1994; K. White et al. 1994), and may not be construed as direct acting HAAs. It is important to keep in mind that the regulatory pathways between the endocrine and immune systems are complex. Alterations in thymic structure and function can affect sex-and adrenal-hormone regulation of immunity, as mediated by the various thymic-hypothalamic-pituitary axes (Grossman 1984, 1985, 1989; Grossman et al. 1991). Halogenated Aromatic Hydrocarbon Compounds It has been well documented that HAHs such as TCDD, polychlorinated dibenzofurans (PCDFs), and PCBs, affect immune response, and they appear to affect all functional arms of the immune system (innate immunity and host resistance, cell-mediated immunity, and humoral immunity) (Table 7-1). Laboratory Studies Specific examples of the immunologic effects of PCBs and TCDD on laboratory animals are detailed in Table 7-2. These HAHs cause atrophy of the thymus, the primary lymphoid organ in which stem cells are selected and differentiated into T-cells. Thymic atrophy has been induced in adrenalectomized animals (Vos and Luster 1989; Lundberg 1991). HAHs cause thymic involution and decrease the number of colony-forming stem cells in animals-and these effects are more dramatic when exposures occur either perinatally or postnatally (Lundberg et al. 1990; Holladay et al. 1991; Lundberg 1991; De Waal et al. 1992: De Heer et al. 1994). This suggests that HAHs target the developing immune system (Fine et al. 1990). Because TCDD directly affects the thymic cortical epithelium, it has been hypothesized that the hormonal factors necessary for lymphocyte maturation are not produced and that thymocytes are pushed into premature terminal differentiation (Greenlee et al. 1985; Lundberg et al. 1990). Exposure to HAHs decreases cell-mediated immune (CMI) responses against bacteria and viruses. This is shown in the decreased host resistance reported in Table 7-1. Treatment with HAHs before immunization with sheep red blood cells—a T-dependent antigen—results in dose- and structure-dependent suppression of immune response in mice (Silkworth et al. 1986; Davis and Safe 1988, 1990; Dickerson et al. 1990; Kerkvliet et al. 1990; Tomar and Kerkvliet 1991). TCDD also has been shown to suppress delayed-type hypersensitivity (Vos and Luster 1989) and to suppress generation and lytic activity of cytotoxic T-cells incontinue

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Page 189 a dose- and strain-dependent manner (Clark et al. 1981. 1983: Nagarkatti et al. 1984). The suppression of cytotoxic T-cells could be related to a concurrent increased number of T-suppressor cells and increased suppressor activity (Clark et al. 1981, 1983; Holsapple et al. 1986); however, that hypothesis is the subject of controversy. Although the mechanism by which HAHs alter CMI responses is unknown, studies have shown that HAHs affect these responses without decreasing T-cell proliferation, IL-2 production, or the number of IL-2 receptors (Dooley et al. 1990). There is evidence that TCDD targets activated lymphocytes rather than resting cells and that TCDD specifically inhibits the activation of antigen-specific T-cells (Dooley et al. 1990; Lundberg et al. 1992). HAHs have been shown to affect humoral immune response. This response is characterized by B-cell antibody production, and it requires B-cell interaction with T-cells and interleukins, which are necessary for B-cell activation and differentiation. B-cells produce antibodies to specific antigens presented by T-cells (T-dependent antigen) or to antigens that cross-link surface immunoglobulins on the B-cell membrane (T-independent antigen). Exposure to HAHs followed by immunization with either T-dependent or T-independent antigens results in a dose- and structure-dependent decrease in antibody production without affecting B-cell proliferation (Davis and Safe 1988, 1990; Kerkvliet et al. 1990; Holsapple et al. 1991; Harper et al. 1995). Although the mechanism by which HAHs suppress the humoral immune response is unknown, it appears that HAHs act by means of the Ah receptor (Silkworth and Grabstein 1982; Lubet et al. 1984; Silkworth et al. 1984, 1986; Kerkvliet et al. 1985, 1990: Davis and Safe 1988. 1990; Howie et al. 1990; Tomar and Kerkvliet 1991; Howie 1992) and that immunotoxic potency correlates with binding affinity. However, it has been reported that components of immunosuppression induced by some HAHs act independently of the Ah receptor (Howie et al. 1990; Kerkvliet et al. 1990: Howie 1992). Field Studies An observational study was conducted between 1992 and 1994 to determine whether contaminant-associated immunosuppression occurs in prefledgling Caspian terns and herring gulls of the Great Lakes (Grasman et al. 1996). The phytohemagglutinin skin test for T-cell mediated immunity was conducted on 3-wk-old chicks at colonies distributed across a broad gradient of organochlorine contamination (primarily PCBs). In both species, there was a strong exposure-response relationship between organochlorines and suppressed T-cell-mediated immunity. Suppression was most severe (30-45%) in colonies in Lake Ontario and Saginaw Bay for Caspian terns and herring gulls, and in western Lake Erie for herring gulls. Although there were significant differences in total antibody and IgG titers among sites, there was no consistent exposure-response relationship with organochlo-soft

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Page 190 TABLE 7-1 Effects of Halogenated Aromatic Hydrocarbons (HAHs) on Immunity HAH Effect Reference Innate Immunity and Host Resistance TCDDa Reduced survival rates of mice infected with influenza virus, herpes simplex Thigpen et al. 1975: Clark et al. 1983; House et al. 1990   type II virus, and Salmonella bern or S. typhimurium   TCDD Decreased serum complement C3 and decreased resistance to Streptococcus White et al. 1986   pneumoniae infection in mice   TCDD In utero exposure to mice significantly increased progeny mortality after infection Luster et al. 1980   with Listeria monocytogenes   TCDD Longer duration of parasitic infection in mice exposed to Plasmodium yoelii Tucker et al. 1986 TCDD Persistent infection in mice treated with Trichinella spiralis Luebke et al. 1994 Cell-Mediated Immunity TCDD Thymic involution and decrease in the number of colony-forming stem cells in Lundberg et al. 1990; Holladay et al. 1991; Lundberg   rats and mice; effects were more dramatic when exposures occurred 1991; De Waal et al. 1992; De Heer et al. 1994   perinatally or postnatally   TCDD Dose- and structure-dependent suppression of immune response in mice after Silkworth et al. 1986; Davis and Safe 1988, 1990; PeCDFb immunization with sheep red blood cells Dickerson et al. 1990; Kerkvliet et al. 1990; Tomar TCDFc   and Kerkvliet 1991 PCBd     HpCDFe     (table continued on next page)break

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Page 191 (table continued from previous page)break HAH Effect Reference TCDD Enhanced immune response in rats to sheep red blood cells Smialowicz et al. 1994 TCDD Suppressed delayed-type hypersensitivity reactions to tuberculin and oxazalone Vos and Luster 1989   in guinea pigs and mice, as well as graft vs. host reactivity in mice and rabbits   TCDD Suppressed generation and lytic activity of cytotoxic T-cells in dose- and strain- Clark et al. 1981, 1983; Nagarkatti et al. 1984   dependent manner   TCDD Altered lymphocyte populations in marmosets Neubert et al. 1992 TCDD Humans exposed to TCDD showed an increased frequency of anergy for Hoffman et al. 1986   delayed-type hypersensitivity responses to the following recall antigens: tetanus,     diphtheria, Streptococcus, tuberculin, Candida, Proteus, and Trichophyton.   PCB PCB-exposed children had nonsignificant decrease in CD4+:CD8+ T-cell ratio Lan et al. 1990   compared with nonexposed children   Humoral Immunity TCDD Exposure to HAHs followed by immunization with Davis and Safe 1988, 1990; Kerkvliet et al. 1990; PeCDF either T-dependent antigen (SRBC) or T-independent Holsapple et al. 1991 TCDF antigen trinitrophenyl-lipopolysaccharide caused dose-   PCB and structure-dependent decrease in antibody production     without affecting B-cell proliferation   a TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. b PeCDF, 1,2,3,7,9-pentachlorodibenzofuran. c TCDF, 2,3,7,8- or 1,3,6,8-tetrachlorodibenzofuran. d PCBs, polychlorinated biphenyls. e HpCDFs, heptachlorodibenzofurans. SOURCE: Adapted from Harper 1995.

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Page 192 TABLE 7-2 Laboratory Studies on Halogenated Aromatic Hydrocarbons Concentration Species Effect Reference Polychlorinated Biphenyls (PCBs) 104-464 mg/kg Mouse Murine splenic PFCa response to SRBCb was functionally inhibited in a dose- Davis and Safe 1989 Aroclor 1260, 1254.   dependent fashion: higher chlorinated PCB preparations were more potent   1248, 1242. 1016.       1232       25 mg/kg/dc Mouse Mice fed PCBs for 6 wk had increased mortality, possibly due to the immuno- Loose et al. 1977 Aroclor 1242   suppressive effects of PCBs on humoral immunity: PFC response to SRBC       antigen by spleen cells was significantly reduced: there was a reduction in       serum IgG1, IgA, and IgM; significant reduction in secondary immune response   5 µg/kg PCB Chick Thymic and spleen involution: marked depletion of lymphocytic cell types in McKinney et al. 1976     both organs   4 µg/kg egg PeCBd Chick Inhibition of lymphoid cell development in the thymus and bursa Andersson et al. 1991 50 µg/kg egg TCBe       300 µg/kg egg HCBf       25-100 ppm Duck Increased mortality following challenge with duck hepatitis virus Friend and Trainer 1970 Aroclor 1254       5-80 µg/kg/d Rhesus monkey Monkeys fed Aroclor 1254 for 23 or 55 mo showed dose-dependent Tryphonas et al. 1989, Aroclor 1254   suppression of anti-SRBC antibody production, no effect on antipneumococcus 1991 a,b     antibody response; at 80 g, decrease in T-helper cell population and increase       in T-suppressor cell population with no change in the mitogen-activated       lymphocyte stimulation: at 55 mo. increase in natural killer cell activity and in       thymosin a-l. but not B-4 or tumor necrosis factor   (table continued on next page)break

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Page 193 (table continued from previous page)break Concentration Species Effect Reference 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) 5-50 µg/kg Mouse Dose-dependent alteration in T-lymphocyte development Lundberg et al. 1990 100 µg/kg Mouse Depletion of cells from thymus and spleen Pung et al. 1984 40 µg/kg Mouse Depletion of thymic cells only; impaired antibody response to antigens SRBC Pung et al. 1984     and trinitrophenyl-Brucella abortus   4 µg/kg Mouse Thymic cell depletion; delayed hypersensitivity response to oxazolone depressed Pung et al. 1984   0.00125 µg/kg/dc Rhesus monkey Monkeys fed TCDD for 4 yr showed selective increase in CD8+ T-cells and Hong et al. 1989       decrease in CD4+ T-cells, no apparent significant effect demonstrated on T-cell       function; natural killer cell activity appeared normal; offspring had significant       increases in generation of antitetanus toxoid antibody production       postimmunization correlated with TCDD tissue concentrations   10 ng/kg Marmoset Marmosets injected with TCDD showed decreased circulating helper-inducer Neubert et al. 1990     T-cells and increased suppressor-inducer T-cells, no change in total number of       T-cells; no-observed-effect level was 3 ng/kg   1.5 ng/kg/wk Marmoset Marmosets treated for 3 wk had depressed helper-inducer T-cells Neubert et al. 1992 a PFC, plaque-forming cell. b SRBC, sheep red blood cell. c Estimated dose based on general assumptions of weight and feed intake. d PeCB, 3,3',4,4',5-pentachlorobiphenyl. eTCB, tetrachlorobiphenyl. fHCB, hexachlorbenzene.

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Page 194 rines. In 1992, altered white blood cell numbers were associated with elevated organochlorine concentrations in Caspian terns but not herring gulls. Although the identity of the specific organochlorine(s) responsible for the suppression of T-cell-mediated immunity could not be determined, the researchers noted that PCBs were the most closely associated with immunosuppression. Field studies of the immunologic effects of HAHs also have been conducted with seals, whales, and dolphins. de Swart et al. (1994, 1996) reported that innate and acquired immune responses were functionally impaired in harbor seals fed herring from PCB-contaminated waters (Baltic Sea) for 126 wk. The estimated intake of PCBs was 1,460 µg/d. Ross et al. (1995) reported similar findings in captive harbor seals fed fish from the PCB-contaminated Baltic Sea. These seals demonstrated impaired ability to mount a delayed hypersensitivity response when challenged with ovalbumin, and they generated 37% less antibody to ovalbumin after antigen challenge than did seals fed fish from the relatively uncontaminated Atlantic Ocean. In exposed seals, the combined concentration of mono-PCB (International Union of Pure and Applied Chemistry (IUPAC) numbers 118, 156, and 189) and diortho-PCB (IUPAC number 180), measured in nanograms of toxic equivalent per kilogram of lipid, was 140.0; it was 35.5 in unexposed seals. De Guise et al. (1994, 1995) studied beluga whales living in the highly contaminated St. Lawrence estuary of Quebec, Canada, and compared them with belugas living in the much less contaminated arctic. They observed that belugas from the St. Lawrence had numerous severe and disseminated infections caused by mildly pathogenic bacteria. They suggested that the generalized immunosuppression was caused by organochlorine contamination. In addition, 75 tumors have been reported in whales worldwide, 28 (37%) of which were found in 18 St. Lawrence beluga whales. The researchers tentatively concluded that this could result from depression in immunosurveillance caused by exposure to environmental contaminants or carcinogens, or both. However, because the reported results were obtained through highly selective (nonrandom) sampling, selection bias might also skew these conclusions. In a study of 15 bottlenose dolphins along the west coast of Florida, peripheral blood lymphocyte responses to Concanavalin A (ConA) and phytohemagglutinin were determined in vitro and compared by regression analysis with contaminant concentrations in whole blood from five of the dolphins (Lahvis et al. 1995). Reduction in ConA-induced lymphocyte responses was correlated with increasing whole blood concentrations of tetrachloro-PCBs (1-18 ng/g), pentachloro-PCBs (4-44 ng/g), hexachloro-PCBs (13-322 ng/g), heptachloro-PCBs (7293 ng/g), and octachloro-PCBs (2-81 ng/g), and similar correlations were also found with DDT (see below). Immunosuppression caused by exposure to environmental contaminants also could account for the severity and extent of morbilli-virus epizootics observed among seals and dolphins (de Swart et al. 1995). However, a direct cause-and-effect relationship has not been proven.break

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Page 195 Human Studies Alterations in immune responses caused by exposure to HAHs have been documented in a few human studies. Lu and Wu (1985) describe the acnegenic and hepatotoxic effects in residents of Yu-Cheng, Taiwan, who ingested high concentrations of PCBs that were accidentally leaked into rice oil. The resulting effects were related primarily to increased respiratory infection; decreased serum concentrations of IgA and IgM; decreased CD4+ T-cells and increased CD8+ T-cells; suppressed dermal delayed hypersensitivity responses to a combination of streptokinase and streptodormase and to tuberculosis antigens; and augmentation of the in vitro lymphocyte mitogen stimulation to phytohemagglutinin (PHA) and pokeweed mitogen (PWM), but not to ConA. Average blood concentrations of PCBs in the affected individuals were 89 + 6.9 ppb. In a study of Wisconsin infants whose mothers ate PCB-contaminated fish, maternal serum PCB levels were positively associated with the number and type of infectious illnesses, such as colds, earache, and flu symptoms, that occurred in infants during the first 4 mo of life (Smith 1984). The authors concluded that prenatal exposure to PCBs was the cause of the increased infections. However, as noted by Swain (1991) in a critique of the study, these results should be interpreted carefully because blood concentrations of PCBs were only measured after birth and not during pregnancy. In a study of Dutch infants, 105 breast-fed and 102 formula-fed infants were evaluated from birth until 18 mo to determine whether prenatal and postnatal exposure to background concentrations of PCBs and dioxins had an effect on the incidences of rhinitis, bronchitis, tonsillitis, and otitis (Weisglas-Kuperus et al. 1995). Humoral immunity was also measured by detecting antibody levels to mumps, measles, and rubella as a result of vaccinations. Prenatal exposure was estimated by PCBs in maternal blood and the total toxic equivalent (TEQ) level in breast milk (measured as pg TEQ/g milk fat), and postnatal exposure was calculated as a product of the total TEQ level in breast milk multiplied by the weeks of breast feeding. Umbilical cord and venous blood was taken from a subgroup of 55 infants at 3 and 18 mo for white blood cell counts and immunologic marker analysis. No relationship was found between pre- and postnatal PCB/dioxin exposure and upper or lower respiratory symptoms or humoral antibody production. However, higher prenatal and postnatal exposures to PCBs/ dioxins were associated with lower monocyte and granulocyte counts at 3 mo, and increases in the total number of T-cells and in the number of cytotoxic T-cells were observed at 18 mo. Recent studies of Inuit people exposed to organochlorines in their diet via sea-mammal fat have reported serum lipid concentrations of 4.1 mg/kg lipids PCBs and 184.2 ng/kg lipids 2,3,7,8-TCDD (Ayotte et al. 1997). Health risk assessments for newborns in these populations indicate a correlation between PCB/dioxin exposures in breast milk and suppressed levels of white blood cellscontinue

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Page 196 in infants (Ayotte et al. 1996). The breast milk of Inuit women contained 7 times more PCBs (sum of the PCB congeners = 1,052 ng/g, lipid) than the milk from women from urban, industrialized areas south of Quebec (Dewailly et al. 1993b). Researchers are investigating the possible connection between unusually high rates of infectious disease, particularly acute ear infections, among Inuit children and exposure to PCBs. Such studies must be interpreted carefully because comparisons of organochlorine concentrations over time are unreliable. Webb et al. (1989) found that humans exposed to TCDD had increased CD8+ T-lymphocyte populations; no change in CD4+ T-cells; no change in lymphocyte response to the mitogens ConA, PHA, and PWM; no change in cytotoxic T-cells; and increased serum IgA. Of the 41 individuals studied, 16 had TCDD concentrations below 20 ppt in their adipose tissue, 13 had concentrations of 20-60 ppt, and 12 had concentrations above 60 ppt (the maximum was 750 ppt). Dichlorodiphenyltrichloroethane DDT has been reported to possess estrogenic and antiandrogenic properties (Kupfer and Bulger 1980), supporting the hypothesis that it acts by binding to steroid receptors in immunologic target tissues. Laboratory studies have demonstrated that DDT can alter both the primary and the secondary humoral immune response, immunoglobulin production, splenic plaque-forming cell (PFC) response, histamine concentrations, and mast cell numbers. Specific examples of DDT's immunologic effects in laboratory animals are detailed in Table 7-3. DDT has been demonstrated to trigger some immunologic effector mechanisms in animal and bird models (Barnett and Rodgers 1994). In studies of harbor seals, a diet of DDT-contaminated fish from the Baltic Sea was shown to impair immune response, as measured by delayed hypersensitivity in the skin to ovalbumin and in vitro lymphocyte assays (de Swart et al. 1994; Ross et al. 1995). In a study of bottlenose dolphins, Lahvis et al. (1995) reported that ConA mitogen assays of peripheral lymphocytes demonstrated a correlation between reduced immune response and increasing concentrations of p,p'-DDT (0-24 ng/g) and p,p'-DDE (13-536 ng/g). l,l-Dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) is a metabolite of DDT. It has been suggested that, with elevated concentrations of these contaminants, a reduction in immune response can be correlated with an increased incidence of infection (Svensson et al. 1994). Overall, the data are suggestive of DDT-mediated immunosuppression in animals and birds being detected at about 10 mg per kilogram of body weight (BW) per day. Additional laboratory studies are needed to identify the functional parameters and immunologic effector cells involved in DDT-mediated immunotoxicity. Until human data are available for comparison, no conclusions can be made about the effects of DDT on the human immune system.break

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Page 199 TABLE 7-4 Laboratory Studies on Endrin and Dieldrin Concentration Species Effect Reference Endrin 3, 4.5, 6 mg/kg Rat Enhanced nitric oxide production by peritoneal macrophages at 24 and 48 hr Akubue and Stohs 1992 4 mg/kg Mouse 3-fold increase in hepatic mitochondrial lipid peroxidation; increase in reactive Bagchi et al. 1993a,b     oxygen species by peritoneal macrophages   Dieldrin 10 M Rat cells Significant stimulation in vitro of polymorphonuclear neutrophils to release Hewett and Roth 1988     superoxide; dependent on the presence of extracellular calcium   0.65 mg/kg/d Mouse After infection with Leishmania, Kupffer cells stimulated to generate soluble Loose 1982     factor that stimulated T-suppressor cell activity in mice treated for 10 wk   18-36 mg/kg Mouse Intraperitoneal injection prolonged recovery from hepatitis infection; depressed Krzystyniak et al. 1985,     production of antiviral IgG Ab 1986 6 mg/kg Mouse Dysfunction of cellular cooperation during the induction phase of the immune Bernier et al. 1987     response with suppressed production of anti-sheep red blood cells IgM and IgG,       and anti-lipopolysaccharide IgM   0.065 mg/kg/d Mouse Decreased antigen processing by alveolar macrophages observed in mice fed Loose et al. 1981     dieldrin for 2 wk   36 mg/kg Mouse Lymphoid cells had strong but transient inhibition of the mixed-lymphocyte Hugo et al. 1988     reaction: mitogen response unaffected in mice administered intraperitoneal       dieldrin   16.6 mg/kg Mouse Inhibition of mixed-lymphocyte reaction Fournier et al. 1988

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Page 200 TABLE 7-5 In Vitro and Laboratory Studies on Lindane Concentration Species Effect Reference In Vitro Studies 0.2-20 µg/mL Human Partial elimination of cell growth at 0.2g µg/mL: complete elimination of all cell Parent-Massin et al. progenitor cells   growth at 2-µ20 g/mL 1994 0.2-200 µ/mL Rat GM-CFUa 0.2 and 2 g/mL significantly stimulated cell growth: 20 and 200 g/mL Parent-Massin et al.   cells significantly reduced, but did not completely eliminate, growth 1994 60, 80, 100 ppm Amoeba Phagocytic activity of labeled Escherichia coli inhibited, possibly because of Gayatri and Chatterjee     alterations in receptor-mediated mechanisms 1993 Laboratory Studies 10. 100 mg/kg Mouse Alteration in cellular and humoral immunity in pups: pups from dams exposed Das et al. 1990     prenatally to 10 mg/kg displayed significantly increased delayed       hypersensitivity to SRBCb; 100 mg/kg significantly impaired delayed       hypersensitivity response to SRBC. At 10 mg/kg. mitogen response of spleen       cells was 2-fold greater for ConAc and 8-fold greater for LPSd: PFCe response       was 2-fold greater. At 100 mg/kg. no effect on mitogen or PFC response   0.012-1.2 mg/kg Mouse Dose-dependent initial stimulation in cell-mediated and humoral immunity. Meera et al. 1992     followed by suppression. in mice fed lindane for 24 wk: no apparent effect on       macrophage function   0.012-1.2 mg/kg Mouse Biphasic effect of lindane corroborated; enhanced calcium uptake during initial Meera et al. 1993     stimulation, return to normal, and suppression in mice fed lindane for 24 wk   15 mg/kg/d Mouse After oral immunization with SRBC. a 2-fold increase in the titers of anti- Andre et al. 1983     SRBC IgG 2b antibody in mice fed lindane for 1 mo; duration of Giardia       muris infection significantly increased; mice developed systemic anti-Giardia       antibodies more frequently   101 mg/kg Mouse No significant change after 30 d treatment Cornacoff et al. 1988 (table continued on next page)break

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Page 201 (table continued from previous page)break Concentration Species Effect Reference 300 mg/kg/d Mouse 44% reduction in natural killer cell activity, significant depression in lympho- Cornacoff et al. 1988     proliferation as measured by phytohemagglutinin, ConA, and LPS response in       mice fed lindane for 30 d   1.3 mg/L Fish Reduced leukocyte counts; no statistical analyses performed Saxena et al 1992 10, 50, 100 mg/kg/d Fish Fish treated with intraperitoneal injections for 45 d; after injection with Dunier and Siwicki 1994     Yersinisa ruckeri vaccine, slight suppression of antibody secretion observed       with 10 mg/kg, effect magnified at higher doses   100 mg/kg Fish Stimulation of Y. ruckeri-antibody-secreting cells and lymphocyte proliferation Siwicki and Dunier     by nitrogranulogen more efficient after immunosuppression induced by lindane 1994 1 mg/kg Fish Depression of nonspecific immune system observed in fish treated for 30 d; Dunier et al. 1994     decreased phorbol 12-myristate 13-acetate-induced phagocytosis by pronephric       cells, depression lasted 2 wk, returned to normal after 1.5 mo   10, 50, 100 mg/kg/d Fish Fish treated with intraperitoneal lindane for 45 d had depressed B-lymphocyte, Dunier et al. 1994     but not T-lymphocyte, proliferation; B-lymphocytes reduced in the head kidney   10, 50 mg/kg Fish 30 d after single intraperitoneal injection of 10 mg/kg significantly reduced Dunier et al. 1995     Ig+ lymphocytes, decreased proliferation of B-lymphocytes; effects not       observed with 50 mg/kg. Significant modification in sera lysozyme and       ceruloplasmin observed at each dose   a GM-CFU, granulocyte-macrophage colony-forming unit. b SRBC, sheep red blood cell. c ConA, concanavalin A. d LPS, lipopolysaccharide. e PFC, plaque-forming cell.

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Page 202 immune system, including effects on humoral and cell-mediated immunity. Notably, lindane at concentrations of 0.012-10 mg/kg stimulated antibody production; production was inhibited at 100 mg/kg. Mitogen response and delayed hypersensitivity displayed similar biphasic effects (stimulation at 10 mg/kg; inhibition at 100 mg/kg). Although lindane at 1 mg/kg inhibited phagocytosis, higher concentrations were needed to inhibit other nonspecific elements of immunity (10 mg/kg for lysozyme concentrations; 300 mg/kg for natural killer-cell activity). Given the various concentrations of lindane tested, diverse routes of administration, differences in length of treatment, and variety of animal models, the exact sites of action of this pesticide and its mechanisms of action on the immune system remain clouded in speculation. Furthermore, with just one limited in vitro laboratory study on the effects of lindane on human immunity, it is premature to extrapolate broad conclusions about its effects in humans. Chlordane Some studies that use laboratory animal models suggest that exposure to chlordane (also under the trade names Octochlor and Velsicol 1068) leads to moderate immunotoxicity. Specific examples of the effects of this pesticide in laboratory animals are presented in Table 7-6. In inhalation studies, chlordane administered at 1 or 10 mg/m3 for 90 d increased lymphocyte numbers in female rats; in vitro tests that used 10 M chlordane showed mitogenic activity. In addition, prenatal exposure to chlordane was reported to alter immune responses—such as delayed hypersensitivity, macrophage activation, and colony-forming unit activity—in offspring of exposed rats. The majority of studies—regardless of the concentration, route of administration, or time of application—found no histologic or functional changes that could be attributed to chlordane treatment. Very limited information is available on the possible immunotoxic effects of chlordane exposure for humans. McConnachie and Zahalsky (1992) report significant changes in cell-mediated immunity and humoral immunity in humans exposed to chlordane aerosols for 3-15 mo. They report impairment in lymphoproliferation to mitogens ConA and phytohemagglutinin A (PHA) and increased titers of autoantibodies in 11 of the 12 subjects tested. These tests were performed from 4 mo to 10 yr after exposure, implying long-term immunotoxicity for this agent. However, the studies are too limited to support any conclusions about the effects of chlordane on human health. Toxaphene There are few data to describe the immunologic effects of toxaphene in laboratory animal models. In one study (Trottmann and Desaiah 1980), thymus weight was reduced in mice after oral administration of toxaphene at 22.5 and 30 mg/kg/d for 14 d. Koller et al. (1983) observed depressed IgG antibody produc-soft

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Page 203 tion in rats treated with 0.5, 1.5, and 10 mg/kg/d for 6 wk. Allen et al. (1983) treated mice with toxaphene at 15 and 30 mg/kg/d for 8 wk and report depressed IgG antibody production. No effect on delayed hypersensitivity was reported. In a prenatal exposure study (Chernoff et al. 1990), pregnant rats were administered 32 mg/kg/d toxaphene by gavage from the onset of pregnancy until gestation d 8, 12, or 16. Rats were killed at these time points or on d 20 of gestation. Spleen weight was significantly reduced in fetuses from rats sacrificed on d 8, 16, and 20; thymus weight was reduced in fetuses from rats sacrificed on d 8 and 20. The studies above are limited, and additional studies are required before a reliable assessment of the possible immunologic effect of toxaphene on animals or humans can be made. Endosulfan Exposure to endosulfan has been reported to produce immunotoxic changes in nonspecific immunity and in humoral and cell-mediated responses in laboratory animals. Immunologic studies with endosulfan are summarized in Table 7-7. Oral exposure to endosulfan induced immunologic effects at 1-5 mg/kg/d (from 6-22 wk), but inhalation and dermal routes did not. The endosulfan studies are limited, and there is little information on the immunologic effects of this compound in humans. Hexachlorobenzene Exposure to hexachlorobenzene has been reported to produce histologic changes in lymphoid tissue architecture in laboratory animals. Vos et al. (1983) report that prenatal and postnatal exposure to hexachlorobenzene at 4 mg/kg/d in feed enhanced humoral and cellular immune responses in rats. Hexachlorobenzene also promoted accumulation of macrophages in the rats' lungs. The high endothelial venules present in the lymph nodes underwent abnormal proliferation, accompanied by lymphoid hyperplasia in the splenic white pulp, in rats fed this compound at 25-100 mg/kg/d for 3 wk (Vos et al. 1979). Hyperplasia of lymphoid tissue in the stomach has been induced in dogs fed hexachlorobenzene at 6.5-10 mg/kg (1 mg/d) for 12 mo (Gralla et al. 1977). No information is available about the possible immunotoxicity of hexachlorobenzene in humans, and the data from laboratory studies are too limited to support any conclusions about how this compound affects human immune response. Summary and Conclusions There are very few studies of the immunologic effects of human exposure to HAAs, but for some chemicals there are adequate data for laboratory animals.continue

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Page 204 TABLE 7-6 Laboratory Studies on Chlordane Concentration Species Effect Reference Inhalation Studies 5.8, 28.2 mg/m3 Rat In rats treated 8 hr/d, 5 d/wk for 28 d, reduction in thymic weight of females Khasawinah et al. 1989     exposed to 28.2 mg/m3 chlordane   1, 10 mg/m3 Rat In rats treated 8 hr/d, 5 d/wk for 90 d, increased lymphocyte numbers in Khasawinah et al. 1989     female rats; no significant change in thymic weight   1, 10 mg/m3 Monkey No significant changes in lymphocyte numbers and no histologic changes in Khasawinah et al. 1989     either lymph nodes or spleen in monkeys treated 8 hr/d, 5 d/wk for 90 d.   Oral Studies 200 mg/kg Rat No histologic changes in the spleen Truhaut et al. 1974, 1975 16 mg/kg/d Rat No effects on spleen weight or spleen histology in rats fed chlordane for 407 d Ambrose et al. 1953 1.25 mg/kg/d Rat No effects on spleen weight or spleen histology in rats fed chlordane for 2-9 mo Ortega et al. 1957 200 mg/kg Mouse No histologic changes in the spleen Truhaut et al. 1974, 1975 8 mg/kg/d Mouse No evidence of immune dysfunction, although leukocytosis and lymphocytosis Johnson et al. 1986     were present in mice administered chlordane by gavage for 14 d   1200 mg/kg Hamster No histologic changes in the spleen Truhaut et al. 1974. 1975 (table continued on next page)break

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Page 205 (table continued from previous page)break Concentration Species Effect Reference 4. 8 mg/kg/d Mouse Offspring from dams fed chlordane for 18 d during gestation had depressed in Barnett et al. 1990a,b     vitro in GM-CFUa and SCFUb; viability of bone marrow cells unaffected; no       effect on GM-CFU or SCFU activity in dams   0. 16 8.0 mg/kg/d Mouse Offspring of dams fed 8.0 mg/kg/d through d 19 of gestation had depression Spyker-Cranmer et al.     in delayed hypersensitivity to oxazolone; no difference in humoral immunity 1982     as measured by plaque-forming cell assay   4.0, 8.0, 16.0 mg/kg/d Mouse Offspring from pregnant mice fed 8 and 16 mg/kg/d through d 19 of gestation Barnett et al. 1985a,b     had significant depression in delayed hypersensitivity responses to oxazolone; Menna et al. 1985     delayed hypersensitivity response to influenza type A depressed; antiviral       antibody titers significantly increased in female offspring only   8 mg/kg/d Mouse Offspring from mice fed chlordane for 18 d during gestation had significant Theus et al. 1992     decrease in 5'-nucleotidase response: macrophages expressed advanced stage       inflammatory responses   Other Studies       67 mg/kg/d Guinea pig Attempts to generate dermal hypersensitivity with chlordane over 90 d Datta et al. 1977     unsuccessful; these animals were not presensitized to chlordane before dermal       application   10, 80 µM Monkey cells In vitro T-cell mitogenic response modified in peripheral blood mononuclear Chuang et al. 1992     cells; at 10 µM, chlordane acted as a T-cell mitogen in the absence of       conventional mitogens: 80 µM completely impaired T-cell function   a GM-CFU, granulocyte-macrophage colony forming unit. b SCFU, spleen colony forming unit.

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Page 206 TABLE 7-7 Studies on Endosulfan Concentration Species Effect Reference Inhalation Studies 2 mg/m3/d Rat Rats exposed for 6 hr/d, 5 d/wk for 21 or 29 d showed no histologic change in Hoechst 1984a     lymph nodes, thymus, spleen   Oral Studies       1, 2, 3, 5 mg/kg/d Rat Significantly depressed humoral response, specifically in serum IgG and IgM Banerjee and Hussain     circulating gammaglobulin fraction, in rats fed endosulfan for 6-22 wk 1986, 1987 1, 2 mg/kg/d Rat Spleen weight reduced in rats fed 1.0 mg/kg for 22 wk; macrophage migration Banerjee and Hussain     inhibition and leukocyte migration inhibition response significantly reduced 1986, 1987     dependent on dose   7.3 mg/kg/d Mouse Male mice fed endosulfan for 13 d had significantly reduced spleen weight Hoechst 1984b Dermal Studies 597 mg/kg/d Guinea pig No measurable sensitization observed in guinea pigs treated for 6 hr/d, 3 d/wk Hoechst 1983     for 3 wk   81 mg/kg/d Rat No effect on thymus weight in male rats treated for 6 hr/d, 5 d/wk for 30 d Hoechst 1985 27 mg/kg/d Rat No effect on thymus weight in female rats treated for 6 hr/d, 5 d/wk for 30 d Hoechst 1985 1,000 mg/kg/d Rat Spleen weight reduced in I male rat after exposure to chlordane for 6 hr, 5 d/wk Hoechst 1989 1% solution Human No skin sensitivity to endosulfan in 48 hr closed patch test of 14 farm workers Schuman and Dobson       1985

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Page 207 Animal studies have identified immunotoxic and immunomodulatory effects. The most extensively studied compounds are the HAHs. Laboratory studies have shown that HAHs affect the functional arms of the immune system. Field studies of birds show a strong exposure-response relationship between organochlorines and immune suppression. Experimental studies have also shown that innate and acquired immune responses were impaired in seals fed fish from the contaminated Baltic Sea. This immunosuppression is believed to be the reason for increased incidences of bacterial and viral infections in seal populations found in contaminated waters. There have only been a few studies of the effects of HAAs in humans, but the results of laboratory and wildlife studies suggest that HAAs have the potential to affect human immune functions. Certainly, additional clinical immunologic end points must be studied. As noted by Kerkvliet and Burleson (1994) ''massive retrospective studies on poorly defined exposure groups cannot be justified to try to 'prove' that immune modulation has occurred in these people." The authors state that "research must focus on the definition of sensitive end points (i.e., biomarkers) of immune dysfunction in humans. . . . In particular it is important to determine in animal models how well changes in immune function in the lymphoid organs (e.g., spleen and lymph nodes) correlate with changes in the expression of lymphocyte subset/activation markers in peripheral blood. Until such correlations are established, the interpretation of changes observed in subset/activation markers in human peripheral blood lymphocytes in terms of health risk will be limited to speculation." HAHs are thought to act through Ah receptor binding, but some components of HAH-mediated immune suppression could function through other, independent mechanisms. The data available on DDT suggest that DDT mediates immunosuppression in laboratory animals and in birds. Certainly, DDT possesses estrogenic properties, and it could act by binding to steroid receptors to modulate immunity. Chlordecone also could modulate immunity through steroid-receptor-binding pathways. Studies of endrin, aldrin, and dieldrin are limited, but the immunologic effects reported for these chemicals in laboratory studies appear to involve macrophage processing of antigen. Reported effects from lindane encompass both the specific and the nonspecific arms of the immune system, and in laboratory animals such responses have included effects on humoral and cell-mediated immunity. Exposure to endosulfan in laboratory studies also has produced immunotoxic effects on nonspecific immunity and altered humoral and cell-mediated responses. In studies of chlordane, moderate immunotoxicity has been observed in laboratory animals, but most studies have not identified histologic or functional changes. Histologic changes in lymphoid tissue architecture were found in laboratory animals after exposure to hexachlorobenzene. There is little information available on the immunologic effects of toxaphene. Generally, the available data neither support nor refute the premise that thecontinue

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Page 208 actions of HAAs is mediated either directly or indirectly through endocrine pathways. It can be stated with some degree of certainty that some HAAs affect one or more aspects of immune function, at least in animal models. Field studies, especially in marine mammals, generally support this view, although there is intrinsic uncertainty for such studies because the conditions of exposure to the environmental contaminants responsible are not known and mixtures of the compounds are not always clearly defined. In human studies, the cause-and-effect relationship between HAAs and immunotoxicity is not clear cut. Thomas (1995) considers all the available data on Great Lakes residents exposed to potentially immunotoxic agents through the food chain and concludes that, based on "uncertainties with regard to exposure levels, predictability of tests, suitability of the animal models, and immune reserve . . . there is no definite evidence as yet that environmental [exposure] to these xenobiotics poses a significant threat to the human immune system." In the few human studies available, exposure to mixtures, extended delays between time of exposure and performance of immunologic tests, and the effects of other confounding variables—age, sex, lifestyle, underlying disease—all tend to limit support for any definitive conclusion. In addition, immunosuppressive effects of background exposures have not been determined. Thus, although animal studies suggest that HAAs can cause immunologic effects, underlying mechanisms are not clear. It is also unclear whether they have similar effects on the human immune system. Recommendations Based on the committee's review of the extensive laboratory animal data on immunologic effects of HAAs, as well as the limited information from wildlife and human studies, the following are recommended: —Comprehensive epidemiologic studies that evaluate a variety of health effects, including immunologic effects, of human populations suspected of being affected by HAAs should be initiated. Especially needed are studies of cohorts established either through registries or directed effort to assess the prevalence of autoimmune problems in offspring whose mothers were exposed during pregnancy. To address the potential problem of measuring exposure in a case-control or cohort design, it is suggested that populations known to have been heavily exposed to HAAs (such as the Seveso population) be used for cohort studies. Ideally, such cohorts should be followed throughout their lifetime. Epidemiologic studies should use clinically relevant immunologic assays, such as those for monitoring concentrations of circulating antibodies to thymus-dependent antigens; antigen test banks to monitor delayed-hypersensitivity skin reactions; quantitative lymphocyte subclass identifications; in vitro measurementscontinue

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Page 209 of lymphocyte cytokine production and possibly mitogenic responsiveness: and the lytic action attributed to cytotoxic lymphocytes and natural killer cells in exposed populations to clarify the relationship between HAA exposure and human health. —Because much of the available immunologic laboratory data on HAAs is on chemicals that have been regulated and, in most cases, are no longer used in the United States, future studies should focus on chemicals that are being used, such as endosulfan and lindane.break