The comparative immunotoxicity of five selected compounds following developmental or adult exposure, 2006
It is well established that human diseases associated with abnormal immune function, including some common infectious diseases and asthma, are considerably more prevalent at younger ages. Although not established absolutely, it is generally believed that development constitutes a period of increased immune system susceptibility to xenobiotics, since adverse effects may occur at lower doses and/or immunomodulation may be more persistent, thus increasing the relative risk of xenobiotic exposure to the immunologically immature organism.
To address this issue, a brief overview of immune maturation in humans is provided to demonstrate that functional immaturity alone predisposes the young to infection. Age-dependent differences in the immunotoxic effects of five diverse compounds, diethylstilbestrol (DES), diazepam (DZP), lead (Pb), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and tributyltin oxide (TBTO), which have undergone adult and developmental immunotoxicity testing in rodents, are then reviewed, as are human data when available. For all five chemicals, the developing immune system was found to be at greater risk than that of the adult, either because lower doses produced immunotoxicity, adverse effects were more persistent, or both.
Between 5 and 10 million pregnant women were given diethylstilbestrol (DES), a potent synthetic nonsteroidal estrogen, between 1938 and 1971 to prevent premature delivery or pregnancy loss. Its use was terminated when a rare form of reproductive system cancer was found in female offspring of DES-exposed mothers. Male and female reproductive systems malformations have been reported in children of treated women, as has anecdotal evidence of immune system dysfunction. DES was also used to increase weight gain in livestock, although this use is no longer permitted in most countries.
Effects on the Immune System in Humans
Both female and male children of DES-exposed mothers report a higher incidence of autoimmune diseases and asthma (Baird et al., 1996). In general, these diseases are considered to be the result of inappropriate immune system responses, or possible loss of homeostatic control, instead of immune system suppression.
Effects on the Immune System in Rodents
In utero exposure
Luster et al. (1978b) reported that a single injection of 0.1 mg DES/kg body weight on gestational day (GD) 16 did not affect the antibody response to the T-cell-dependent antigen, sheep red blood cells (SRBC), when evaluated in 7-wk-old male and female offspring of Swiss-Webster mice. The T-independent IgM response of female offspring to bacterial lipopolysaccharide (LPS) was suppressed by DES, but was similar to control responses when females were
immunized for a second time. In marked contrast, the male offspring response to LPS immunization was enhanced after both first and second immunizations, an effect attributed to the stimulating effect of estrogen on the antibody response to LPS. Delayed-type hypersensitivity responses (DTH) were suppressed in female, but not in male, offspring, even though thymus weights and T-cell responses to polyclonal stimulation were suppressed in both genders (Luster et al., 1979). Further studies suggest that DES targets early precursors of T lymphocytes in the fetal liver, accounting for thymic atrophy and suppression of DTH (Holladay et al., 1993), but not for defects in T-independent responses to LPS of female offspring.
Nonspecific T- and B-cell proliferation was reported to be suppressed in 6-wk-old female NMRI mice given 5 μg DES/d (roughly 2.2 mg DES/kg/d) over postnatal days (PND) 1–5 (Kalland et al., 1979); suppression was still evident at 17 mo of age (normal life span ~24 mo). It is noteworthy that neither estradiol nor corticosterone exposure over PND 1–5 produced long-term suppression, and that lymphocyte proliferation was comparable to control values at 6 wk of age in females exposed to DES over PND 6–10. Lower doses (approximately 4.4, 44, or 440 μg/kg/ d) had no effect on proliferative response. The 5-μg DES/d exposure regimen also decreased NK cell activity in 6- to 8-wk-old female inbred C57Bl/6 (75%↓) and BALB/c (53%↓) mice and in outbred NMRI (28%↓) mice (Kalland, 1980a). NMRI or AKR/J female mice, exposed to 5 μg DES/d over PND 1–5, were also more likely to develop tumors after low dose injection of a known carcinogen (Kalland & Forsberg, 1981). A subsequent paper (Kalland, 1984) reported that, on a per cell basis, NK cells from DES mice were as active as cells from the control group, but that exposure reduced the number of NK cell precursors in the bone marrow. In other words, NK cells from experimental animals were as efficient as those from controls, but a deficiency in NK cell precursors produced functional suppression of NK activity at the whole animal level. The same postnatal exposure regimen (Kalland, 1980a) reduced the T-lymphocyte-dependent antibody response to SRBC by ~60%, and the T-independent response to bacterial LPS by ~40% when examined in 16- to 18-wk-old NMRI mice. Suppression of the T-dependent response was reportedly due to a defect in T-helper cells. DTH responses were likewise suppressed in 6- and 9-mo-old NMRI females exposed to approximately 2.2 mg/kg/d over PND 1–5 (Kalland & Forsberg, 1978). Kalland (1980b) also reported a persistent (at least 6.5 mo postpartum) decrease in the proportion of T cells in the spleens of DES-exposed mice.
Luster et al. (1980) reported suppression of the antibody response to SRBC or LPS, and the DTH to keyhole limpet hemocyanin (KLH), in adult female mice exposed to 2 or 8 mg DES/kg/d × 5 d. The DTH was decreased in mice dosed with DES after, but not before, sensitization with KLH, suggesting that the suppressive effects of DES on DTH were not persistent. Using the same exposure regimen, resistance to bacterial or parasite infection was decreased and tumor incidence in animals challenged with tumor cells was increased at ≥2 mg DES/kg/d (Dean et al., 1980). T-cell-mediated resistance to a nematode infection was suppressed by 5 d of exposure to 0.2 mg DES/kg/d if exposure began on the day of infection; if exposure commenced 5 d before or 3 or 8 d after infection, decreased resistance was only observed at the highest dose (8 mg/kg/d) (Luebke et al., 1984).
Mode(s) of Action
DES is a potent estrogen, and likely affects immune function via the estrogen receptor (ER). Evidence includes similar effects of known estrogens (17β-estradiol) on the immune system of adult and neonatal rodents, blockade of certain immunotoxic effects by pharmacologic antagonism of the ER (Luster et al., 1984), and antagonism of estrogen-mediated immune system effects in mice lacking ERα (Staples et al., 1999). DES appears to target precursor cells in the bone marrow (adults and neonates) and fetal liver (neonates), producing a long-lasting or perhaps permanent reduction in numbers of precursor cells. This defect explains a significant portion of long-lived immunosuppressive effects (e.g., Kalland’s 1984 paper on suppressed NK activity), although the effects of adult exposure also includes damage to the thymic epithelium (Luster et al., 1984). The underlying mechanism of long-term suppression following exposure of the developing immune system to DES is not known, but the default assumption is that a critical cell population is lost to developmental exposure; either this purported population is refractory to estrogen-mediated ablation in adults or repair and recovery mechanisms are present in adults that are lacking in the developing immune system.
There has been no systematic evaluation of persistent DES-mediated immunosuppression in adult animals. Dose-response data are not available for many of the of the developmental exposure studies that revealed persistent effects.
In utero exposure to 0.1 mg DES/kg during the last trimester of pregnancy suppressed T-cell- and B-cell-mediated responses only in female offspring. The gender dependence of effects was remarkable in that T-independent responses in male offspring were enhanced, yet suppressed in females. Exposure during gestation produced effects that persisted into the equivalent of young adulthood. In neonates there appears to be a critical developmental window during PND 1–5, during which exposure to DES produces persistent immune system defects that last well into adulthood or persist for most of the normal life span of the mouse. These effects are among the most persistent reported for any chemical. In adults, immunosuppression occurs at doses similar to those that produce immunotoxicity in developing animals. However, the immune system-related endpoints that have been evaluated over time in exposed adult animals (bone marrow cellularity, thymus weights) recover relatively quickly (Forsberg, 1984). In adults, recovery may occur so quickly that suppression of cell function or resistance to infection may require ongoing exposure to maintain suppression.
Immunotoxicity has been reported at similar doses when exposure occurs during late gestation, early postpartum, or as adults. However, the distinguishing feature of developmental exposure to DES is the persistence of effects, some of which are still apparent in very old mice. In contrast, immune system-related endpoints that have been evaluated (bone marrow cellularity, thymus, weights) suggest that adults recover relatively quickly (Forsberg, 1984).
- The comparative immunotoxicity of five selected compounds following developmental or adult exposure, Journal of toxicology and environmental health. Part B, Critical reviews, NCBI PubMed, PMID: 16393867, 2006 Jan-Feb.
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