2024 Volume 49 Issue 5 Pages 209-218
The immune system is sensitive to many chemicals. Among dioxin compounds, 2,3,7,8-tetrachlorodizenzo-p-dioxin (TCDD) is the most toxic environmental pollutant. The effects of perinatal maternal exposure to dioxins may persist into childhood. However, there have been no reports to date on the effects of exposure to dioxins during infancy, when the immune organs are developing. Therefore, we investigated the effects of TCDD and antigen exposure during lactation on immune function, especially antibody production capacity, in adult mice. Beginning the day after delivery, lactating mothers were orally administered TCDD or a mixture of TCDD and ovalbumin (OVA) daily for 4 weeks, until the pups were weaned. At 6 weeks of age, progeny mice were orally administered OVA daily for 10 weeks, while non-progeny mice were orally administered OVA or a mixture of TCDD and OVA daily for 10 weeks. Production of serum OVA-specific IgG was examined weekly. The amount of TCDD transferred from the mother to the progeny via breast milk was determined by measuring TCDD in the gastric contents of the progeny. A trend toward increasing IgA titer was observed in TCDD-treated mice, and production of IgE was observed only in progeny whose mothers were treated with TCDD and OVA. The results suggest that exposure to TCDD and OVA in breast milk can affect immune function in newborns.
The human immune system is sensitive to the toxic effects of many chemicals, and these health effects can manifest in various ways, such as through allergies, infectious diseases, and carcinogenesis. The immune response, a biological defense system that eliminates pathogens and other non-self factors to protect the body, plays a major role in lymphoid tissues such as the thymus and spleen and grows significantly during infancy and puberty, reaching its maximum during adolescence (Scammon, 1930; Fu and Chaplin, 1999). Therefore, it is assumed that exposure to chemicals and allergens during the fetal and infantile stages of tissues along with functional underdevelopment may cause abnormalities in the maturation, differentiation, and development of lymphoid tissues, which in turn may induce lifelong immune-related diseases through functional decline or hyperfunction. The cause of a disease may be fetal exposure to chemicals or other substances that adversely affect the environment within the mother and breast milk. Furthermore, as a critical point exists in the fetal and neonatal period when sensitivity to homeostatic disruptors is high (Yoshida et al., 2002), it can be inferred that exposure to chemical substances in the fetal period and infancy may be a possible trigger of the above-mentioned diseases.
Because of the hazardous nature of environmental pollutants such as dioxins and brominated flame retardants, emissions and other standards have been set by governments; as a result, the number of reported cases of serious health hazards due to exposure to high concentrations of pollutants, as was the case in the past, is on the decline. Nevertheless, various environmental contaminants continue to be detected in human blood, breast milk, and food (Needham et al., 2011; Kitamura et al., 2005; Ae et al., 2018). In other words, many of the environmental pollutants to which we are exposed on a daily basis are ingested through the diet, and these unintentionally ingested chemicals are absorbed through the intestinal tract, distributed to various organs, and remain in the body for a long period of time through repeated intestinal circulation (Schecter et al., 2006; Muncke, 2009; Costa et al., 2014).
Many environmental pollutants have endocrine-disrupting effects, and among dioxin compounds, 2,3,7,8-tetrachlorodizenzo-p-dioxin (TCDD) is the most toxic, causing a variety of adverse effects, including carcinogenicity, immunosuppression, and reproductive toxicity (Clark et al., 1991; Giri, 1986; Ohsako et al., 2010). TCDD has also been detected in human breast milk and foods (Hooper et al., 1999; Ohta et al., 2004; Yang et al., 2002). One study reported that pregnant mice administered TCDD exhibited teratogenic effects in the pups, such as cleft palate and hydronephrosis (Mimura et al., 1997). Prenatal exposure to polychlorinated biphenyls and dioxins is also reportedly associated with changes in T-cell lymphocytes in healthy infants, and the effects of perinatal maternal exposure may persist into childhood and become associated with higher susceptibility to infection (Weisglas-Kuperus et al., 2000).
Though there have been reports of fetal exposure to dioxins, to date there are no reports on the effects of exposure during infancy, when the immune organs are developing and the impact on the immune response is unknown. The study reported here compared the effects on immune function in mice exposed to dioxin (TCDD) and antigen during lactation and when exposed to these substances in the puberty period. Of particular interest was the potential effect on antibody production capacity as the pups matured into adulthood.
TCDD was purchased from Cambridge Isotope Laboratories (Massachusetts, USA) and dissolved in saline containing 10% Tween-20 and 1% ethanol. All other reagents used in the study were of the highest quality commercially available (Nacalai Tesque, Kyoto, Japan). BALB/c mice (male and female; 5 to 7 weeks old) were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). Upon arrival, the mice were housed at a constant temperature of 23 ± 1.5°C with a 55% relative humidity under a 12-hr light/12-hr dark cycle and provided ad libitum access to standard rodent chow and water. All animal experiments in this study were performed accordance with the applicable guidelines of Setsunan University and approval of its Ethical Use of Experimental Animals Committee.
Exposure to TCDD and ovalbumin (OVA)The exposure and overall experimental design are indicated in Figures 1A, 2A, and 3A. Eight-week-old mice were mated. The day after delivery, the pups were prepared into six mice, and lactating mothers were orally administered 100-µL aliquots of TCDD (0-500 ng/kg/day), OVA (100 µg/mouse) or a mixture of TCDD (0-500 ng/kg/day) and OVA (100 µg/mouse) daily for 4 weeks until weaning (Kakutani et al., 2021). The day after the final dosing, lactating mothers were separated from their progeny. Only if there are two or three females at a time when it is possible to distinguish between males and females, female progeny mice were housed for 2 weeks under normal conditions. At 6 weeks of age, progeny mice were orally administered 100-µL aliquots of OVA (100 µg/mouse) daily for 10 weeks. By contrast, 6-week-old female non-progeny mice (i.e., “normal mice”) were orally administered 100-µL aliquots of OVA (100 µg/mouse) or a mixture of TCDD (0-500 ng/kg/day) and OVA daily for 10 weeks.
Effects of TCDD exposure during lactation. (A) Experimental design. (B, C) Body weights. (B) Lactating mothers the day after delivery were orally administered TCDD for 4 weeks. (C) Six-week-old progeny mice exposed to TCDD via breast milk were orally-administered OVA daily for 10 weeks. Production of (D) serum OVA-specific IgG and (E) fecal OVA-specific IgA in progeny mice. Values are shown as means ± SE (n = 5 [mother] or n = 10-15 [progeny]). *p < 0.05, **p < 0.01, compared with vehicle in the same week.
Effects of TCDD and OVA exposure during lactation. (A) Experimental design. (B, C) Body weights. (B) Lactating mothers the day after delivery were orally administered TCDD and OVA for 4 weeks. (C) Six-week-old progeny mice exposed to TCDD and OVA via breast milk were orally-administered OVA daily for 10 weeks. Production of (D) serum OVA-specific IgG and (E) fecal OVA-specific IgA, and (F) serum OVA-specific IgE in progeny mice. ■ indicates below the detection limit. Values are shown as means ± SE (n = 5 [mother] or n = 10-15 [progeny]). *p < 0.05, **p < 0.01 compared with vehicle in the same week.
Effects of TCDD and OVA exposure in puberty mice. (A) Experimental design. (B) Body weights. Six-week-old normal mice were orally administered TCDD and OVA daily for 10 weeks. Production of (C) serum OVA-specific IgG and (D) fecal OVA-specific IgA in normal mice. Values are shown as means ± SE (n = 10). *p < 0.05, ** p < 0.01 vs. vehicle in the same week.
After the first administration, serum and mucosal secretions (i.e., fecal extracts) were collected weekly. Fecal samples (from mice placed individually in clean bedding) were collected on the same day as blood collection. For the latter, blood was obtained by tail vein bleed into capillary tubes. All weekly-sampled fecal pellets (≈ 100 mg) collected/mouse were suspended in 1 mL phosphate- buffered saline (PBS, pH 7.4) and vortexed at 4°C for 10 min. After centrifugation at 3,000 x g for 10 min, the resulting supernatant (“extract”) was collected. Serum from each blood sample was isolated using standard protocols and frozen at -80°C until analyzed.
OVA-specific antibodies in serum and fecal extracts were titrated using an ELISA (enzyme-linked immunosorbent assay). In brief, wells of a 96-well immunoplate were coated with OVA (100 µg/well) in carbonate buffer (pH 9.6) and then incubated with 4% BlockAce (Yukijirushi Nyugyo Co., Tokyo, Japan) for 2 hr at room temperature. The samples were then serially diluted 10-fold in 0.4% BlockAce diluted with distilled water and incubated in OVA-coated wells for 2 hr at room temperature for determination of IgG and IgA or overnight at 4°C for determination of IgE. After the respective incubation periods, wells were gently rinsed with TBS-T (Tris-buffered saline [pH 7.4] containing 0.05% Tween 20) before each well received a solution of horseradish peroxidase (HRP) -conjugated anti-mouse IgG, IgE, or IgA (at manufacturer-recommended dilution; Southern Biotechnology, Birmingham, AL) in 0.4% BlockAce. The plates were then incubated for 1 hr at RT before the wells were gently rinsed with TBS-T. To detect the presence of the OVA-specific isotypes in the wells, 100 µL of a solution of 200 mg TMB (3,3',5,5'-tetramethylbenzidine peroxide)/mL substrate (Thermo Scientific, Rockford, IL, USA) was added to each and the plate incubated for 20 min at RT. Reactions in each well were then stopped by addition of 100 µL 2 M H2SO4, and absorbance values at 450 nm were determined using an LB 941 microplate reader (TriStar-Berthold, Bad Wildbad, Germany). End- point titer was calculated as the reciprocal of the highest dilution resulting in an optical density value of 0.1 (at 450 nm), after subtracting the optical density in the control wells.
TCDD concentration in breast milkTwenty-four hr after TCDD administration, gastric contents were collected from progeny mice as breast milk and then saponified by the addition of 1 M KOH/ethanol and shaking at room temperature for 2 hr using a mechanical shaker. TCDD was extracted twice with ethyl acetate using 3-mL Bond Elut C18 cartridges (500-mg packing; Agilent, Santa Clara, CA, USA). The resulting extract was then evaporated to dryness, re-dissolved in n-hexane, and fractioned over a KOH silica gel column (Wako Chemicals, Osaka, Japan). The purified extract was evaporated to dryness, re-dissolved in 20 µL of toluene, and analyzed by GC-MS in EI-SIM mode. The concentration of TCDD was determined using an Agilent 7890B coupled to a JEOL JMS-Q1500GC (GC-QMS) operated at a resolving power of 3,000. The MS was operated in electron ionization mode (EI, 70 eV, 50 µA; 250°C). The 504.9696 m/z ion of perfluorokerosene was used as the lock-mass. TCDD analysis was carried out using a DB-5MS column (30 m × 0.25 mm, 0.25 µm film thickness) under the following chromatographic conditions: initial temperature of 100°C held for 1.5 min, increased to 200°C at 20°C/min, then increased to 320°C at 15°C/min. The columns were connected directly to the mass spectrometer ion source (interface temperature 250°C). A 1-µL aliquot of each sample was introduced into the system by split-less injection (injection temperature 250°C, 1 min split-less time).
TCDD was identified through the comparison of the retention time and mass spectra with those of authentic standards. Peak area ratios for (M)+/(M+2)+ were determined from SIM chromatograms. The acceptance criterion was set as −30% to 30% of the ratios observed with authentic standards. TCDD concentrations were corrected via comparison with the recovery of the respective 13C-internal standards. Samples that exhibited high recovery rates (ranging from 60 to 120% relative to the respective internal standards) were used for data collection.
Statistical analysisAll values are reported as means ± standard error (SEM). The statistical significance of differences in means was determined using one-way analysis of variance followed by Dunnett’s multiple comparisons test. Significance was accepted at p < 0.05. All analyses were performed using Prism software (v.10; GraphPad, San Diego, CA, USA).
Most animals that receive a lethal dose of TCDD exhibit sudden weight loss (i.e., systemic wasting syndrome) and die within a few weeks of exposure (Ministry of Health, Labour and Welfare, 1996). It has previously been reported that continued administration of 1 µg/kg BW/day resulted in the death of all mice in the second week of treatment (Kakutani et al., 2021). And, if the animal is affected by a major disorder of the spleen related to lymphocyte production (such as decreased spleen weight), the ability to produce antibodies also decreases. We therefore monitored mice for sudden weight loss and evaluated spleen toxicity by monitoring body weight and spleen weight. Mother mice were exposed to TCDD at concentrations of 50, 100, or 500 ng/kg. Neither progeny mice (Figs. 1B and 2B) nor normal mice (Fig. 3B) exhibited a significant decrease in body weight compared with vehicle-gavaged controls. In addition, there were no differences in spleen wt/body wt (splenic indices) between TCDD-treated and control mice (Table 1). There were also no significant effects on splenic weights of progeny mice exposed to OVA. And, no pups died during the experiment. These observations confirmed that TCDD caused no systemic side-effects or macro-effects on a key immune organ at the concentrations used in this study.
| A, TCDD exposure during lactation | |||
| TCDD administration | Body weight | Spleen weight | Spleen/body weight |
| (ng/kg) | (g) | (mg) | (ratio) |
| 0 | 20.8 ± 0.16 | 117.6 ± 1.79 | 5.66 ± 0.06 |
| 50 | 21.1 ± 0.45 | 120.9 ± 2.95 | 5.74 ± 0.16 |
| 100 | 21.4 ± 0.31 | 119.2 ± 1.89 | 5.57 ± 0.07 |
| 500 | 21.1 ± 0.27 | 117.5 ± 1.36 | 5.58 ± 0.11 |
| B, TCDD and OVA exposure during lactation | |||
| TCDD administration | Body weight | Spleen weight | Spleen/body weight |
| (ng/kg) | (g) | (mg) | (ratio) |
| 0 | 19.4 ± 0.31 | 117.9 ± 1.49 | 6.16 ± 0.14 |
| 50 | 19.7 ± 0.97 | 117.8 ± 1.01 | 6.51 ± 0.31 |
| 100 | 19.8 ± 0.17 | 118.7 ± 1.67 | 5.93 ± 0.10 |
| 500 | 19.8 ± 0.39 | 118.6 ± 1.06 | 6.00 ± 0.08 |
| C, TCDD and OVA exposure in adult mice. | |||
| TCDD administration | Body weight | Spleen weight | Spleen/body weight |
| (ng/kg) | (g) | (mg) | (ratio) |
| 0 | 21.3 ± 0.56 | 113.8 ± 3.72 | 5.36 ± 0.24 |
| 0.5 | 20.6 ± 0.20 | 123.9 ± 4.56 | 6.02 ± 0.26 |
| 5 | 21.0 ± 0.48 | 128.6 ± 7.44 | 6.14 ± 0.38 |
| 50 | 21.1 ± 0.07 | 128.6 ± 8.49 | 6.09 ± 0.38 |
| 100 | 21.4 ± 0.38 | 121.5 ± 9.56 | 5.68 ± 0.48 |
| 500 | 20.4 ± 0.52 | 126.1 ± 7.67 | 6.21 ± 0.47 |
(A) Progeny mice (n = 15) at six weeks old exposed with TCDD for lactation period were orally administrated OVA for 10 weeks. The body weights of the mice were measured every day, and were shown at week 10. (B) Progeny mice (n = 15) at six weeks old exposed with OVA and TCDD for lactation period were orally administrated OVA for 10 weeks. The body weights of the mice were measured every day, and were shown at week 10. (C) Normal mice (n = 10) at six weeks old were orally administrated OVA and TCDD for 10 weeks. The body weights of the mice were measured every day, and were shown at week 10. The data are shown as means ± SE.
According to Scammon's growth curve, lymphoid tissue is in a period of rapid growth during infancy, with maximum growth during puberty (Scammon, 1930). To examine the effects of TCDD exposure on immune function in lactating and puberty mice, the effect on antibody production capacity was examined using the protocol shown in Figs. 1A, 2A, and 3A. When OVA was administered to 6-week-old progeny mice, the antibody titer increased over time, and all progeny mice of mothers treated with TCDD exhibited a concentration-dependent increase in antibody titer (Figs. 1D, 1E, 2D and 2E). In the group of lactational exposed to only TCDD, the IgG titer began to increase starting in the fifth week of OVA administration. In contrast, in the group of lactational exposed to TCDD and OVA, the IgG titer began to increase starting in the third week of OVA administration. On the other hand, when TCDD and OVA were administered to normal mice, a concentration-dependent increase in IgG titer was observed starting at the fifth week of treatment (Fig. 3C). It was suggested that lactational combined lactational exposure to TCDD and OVA, rather than exposure to TCDD alone in lactation or TCDD and OVA in adulthood, affects immune responses, especially antibody production capacity, in adulthood. We infer that these results have biologically and/or clinically significant.
Interestingly, production of IgE was observed from fourth week of treatment only in progeny whose mothers were treated with TCDD and OVA; antibody titer at week 10 were 3.06 ± 0.59. Other doses were below the detection limit. This result means that lactational exposure to TCDD and OVA is more likely to induce allergies in adulthood.
TCDD concentration in progeny miceProgeny were exposed to TCDD via breast milk, but it is unclear how much TCDD they are actually exposed to. Therefore, the exposure (dose) of the progeny was examined using the gastric contents as breast milk (Fig. 4). The TCDD concentration on the day after administration was 571.93 ± 57.04 fg/mg gastric contents and remained essentially stable until 11 days after administration. However, the TCDD concentration decreased significantly after 13 days and continued to decrease. These data indicated that the concentration of TCDD decreased as the mice began to consume feed rather than breast milk.
TCDD concentration in gastric contents. Gastric contents of progeny raised from mothers exposed to TCDD (500 ng/kg/day) were collected as breast milk. Gastric contents were cleaned, and TCDD concentration measured by GC-QMS. Values are shown as means ± SE (n = 5). **p < 0.01 vs. Day 1.
One reason for the rapid increase in the incidence of allergic diseases is presumably that the number of people, including children, who develop allergic diseases is increasing every year due to rapid and significant changes in our environment and increased exposure to environmental pollutants, including chemical substances. Humans are continuously exposed to various environmental pollutants beginning in the fetal period, when the body defense mechanisms are most vulnerable, and continue until death. Exposure of mammalian mothers to chemicals during gestation and lactation reportedly alters the metabolic capacity of the fetus and infant (Kirchner et al., 2010; Hachisuka et al., 2010). The present study examined the immunomodulating effects of exposure of adult mothers to the environmental pollutant (TCDD) during the infancy of progeny, with a particular focus on any impacts on antibody production capacity of the offspring as they matured.
Even though TCDD exposure during the lactation period was negligible compared to exposure during adulthood, exposure to TCDD via breast milk enhanced the immune response to the antigen during adulthood. Interestingly, pups raised by mothers that were treated with TCDD and OVA had higher antibody titers than those raised by mothers that received TCDD alone. This suggests that the transfer of environmental contaminants and antigens to infants via their mother's milk affects the development of the immune system and modulates the antibody-producing capacity of infants when they mature due to prior antigen exposure.
TCDD is persistent and highly accumulative, with a very long biological half-life in humans of 2340 days (7.78 years) (Geyer et al., 2002). Therefore, it is presumed that TCDD ingested through breast milk is retained in the body for a long period of time. Immunotoxicological studies using mice exposed to TCDD have shown dramatic changes in many immune cells, including changes in cytokine profiles (Lai et al., 1997; Kerkvliet, 2009; Ishikawa, 2009; Yamada et al., 2012). It was also reported that oral administration of TCDD causes shifts in the T-helper cell class (TH1/TH2) balance toward a TH1 response (Kakutani et al., 2021). The absence of a significant difference in the weight of immune organs such as the spleen in this study suggests to us that the observed results were most likely due to changes in cytokine profiles and the differentiation potential/population profiles among B- and T-cells. More-detailed studies examining factors such as immune cell populations and circulating cytokines are thus needed in the future.
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor involved in xenobiotic metabolism, cellular proliferation, neurotoxicity, and tumor promotion and differentiation (Ema et al., 1994; Safe, 1986). The AhR also regulates important immune processes in response to endogenous and environmental cues (Quintana and Sherr, 2013; Stevens et al., 2009; Stockinger et al., 2014). Two major groups of AhR ligands have been described (Denison and Nagy, 2003). The classical group exhibits affinity for the AhR and includes environmental contaminants such as halogenated aromatic hydrocarbons, including dioxins. The nonclassical group comprises a number of structurally diverse chemicals that function as relatively weak inducers of CYP1A1 and/or exhibit less affinity for the AhR than TCDD. Most of the known ligands described to date are synthetic environmental contaminants, such as halogenated aromatic hydrocarbons. It is probable that AhR ligands exhibit great structural diversity and are ubiquitous in the environment. It is therefore assumed that the results of the present study would be generally reproducible with regard to exposure to other AhR ligands, although further studies are needed.
The reported AUC0-120 min of OVA is approximately 6 min•µg/mL with oral administration of 50 mg OVA (Matsubara et al., 2008). In lactating mother animals exposed to an aerosol of 0.5% OVA for 20 min, the concentration of OVA in breast milk after 6 hr would be 180 ± 20 ng/mL (Verhasselt et al., 2008). Based on these findings, it is likely that orally administered OVA was transferred to the breast milk in this study as well, but as OVA could not be detected in the gastric contents by Western blot analyses. In general, it is most likely that the concentration in the hosts was in the fg ~ ag level. It has been reported that various food antigen-specific IgE was detected in the serum of 2-6-month-old infants diagnosed with atopic dermatitis before the start of weaning, suggesting that food proteins inoculated by the mother may induce allergic sensitization via a milk (Ito, 2002). In this study, it is inferred that orally administered OVA maintains its antigenic properties, migrates into the blood, and is excreted into breast milk, thereby affecting the immune system of the newborns.
In conclusion, the results of this study suggest that complex exposure to TCDD and OVA in breast milk induces allergic symptoms in newborns by affecting immune function. As TCDD and dietary antigens are reportedly present in human breast milk (Ito, 2002; Ulaszewska et al., 2011; Hirose et al., 2001; Palmer and Makrides, 2006; Vance et al., 2005), further detailed analyses of the mechanism of immune disruption seen here need to be undertaken. How various hazardous agents, including TCDD, act needs defining; that information will be of great help aid in elucidating the onset/exacerbation of allergic reactions caused by exposure to hazardous chemicals.
This study was supported by a Grant-in-Aid for Scientific Research (C) (grant no. 16K09113) from the Japan Society for the Promotion of Science.
Conflict of interestThe authors declare that there is no conflict of interest.
