Tai L. Guo

Genistein and methoxychlor modulate the activity of natural killer cells and the expression of phenotypic markers by thymocytes and splenocytes in F0 and F1 generations of Sprague–Dawley rats

The isoflavone genistein (GE) and methoxychlor (MXC) have been shown to be estrogenic in both in vitro and in vivo experimental systems. The objective of the present study was to evaluate the effects of GE and MXC on the immune system in adult and developing rats and the potential interaction between these compounds in their immunomodulatory actions. Timely pregnant Sprague-Dawley rats were exposed to GE (300 or 800 ppm), MXC (800 ppm), or their combinations in feed starting on day 1 of gestation. The offspring were exposed to these chemicals gestationally and lactationally. Immunological evaluation was performed on postnatal day 22. In F0 females, exposure to GE had no effect on the percentages of thymocyte subsets, but caused a significant decrease in the absolute thymus weight at the 800-ppm dose level. In the spleen, GE did not affect the activity of natural killer cells but induced changes in the percentages of splenic T lymphocyte subsets. Exposure to MXC produced no effect on the immune parameters examined except for a decrease in the percentage of CD4+CD8- thymocytes. Additionally, minimal interaction between GE and MXC was observed. In F(1) males, both GE and MXC decreased the percentage of CD4+CD8- thymocytes, but only GE increased spleen natural killer cell activity. MXC in combination with 300 ppm-GE, but not separately, produced significant decreases in the absolute weights of thymus and spleen. In F1 females, GE decreased the percentage of CD4+CD8- thymocytes, increased the percentage of CD4+CD8+ thymocytes, and decreased the activity of spleen natural killer cells. In contrast, MXC increased the percentages of spleen natural killer cells and CD8+ T cells. Overall, the results demonstrate that both GE and MXC can modulate the immune system with greater effects observed in developing rats. Moreover, male and female rats have differential responses to these compounds. A lack of interaction between these two estrogenic chemicals in modulating these immune parameters indicates that their effects on the immune system might involve other mechanisms in addition to the estrogen receptors.

Contact sensitizing potential of annatto extract and its two primary color components, cis-bixin and norbixin, in female BALB/c mice.

The present studies were performed to examine the contact allergenic effects of an annatto extract (ANT) in female BALB/c mice. ANT at 5-10% induced a greater than threefold increase in lymph node cell proliferation when compared to the control in the LLNA. Moreover, a significant increase in the percent ear swelling at 24h after ANT challenge was observed in the MEST. A significant increase in the percentage of B cells was also observed. To determine which of the two predominant coloring components (norbixin and bixin) in ANT was responsible for the sensitizing effects of ANT, norbixin was subsequently examined, with negative results being observed in both the LLNA and MEST following treatment with norbixin (1-20%). These findings suggested that perhaps bixin was responsible for the positive responses in both the LLNA and MEST following exposure to ANT. Therefore, further studies using a partially purified cis-bixin extract were conducted. Positive responses in both the LLNA and MEST were observed in mice treated with cis-bixin at the concentrations as low as 0.1-0.5%. These results have demonstrated that cis-bixin, but not norbixin, is likely a contact sensitizer and contributes to the contact hypersensitivity effects observed following dermal exposure to ANT in mice.

Genistein Protects Female Nonobese Diabetic Mice from Developing Type 1 Diabetes When Fed a Soy- and Alfalfa-free Diet

The objective of this study was to determine the effects of the phytoestrogen genistein (GEN) on the time of onset and/or the incidence of type 1 diabetes (T1D) in female nonobese diabetic (NOD) mice, when administered GEN by gavage once every day for up to 180 days. Five groups of mice (approximately 24 animals/group; 6–7 weeks of age) were included: naive control, vehicle control (25 mM Na2CO3 in water), and 3 GEN treatment groups (2 mg/kg, 6 mg/kg, and 20 mg/kg). Mice were maintained on a soy- and alfalfa-free diet (5K96) during the study and were monitored for blood glucose changes every week. When compared to the vehicle control, exposure to 2-mg/kg GEN produced significant decreases ranging from 55 to 79% in the total incidences of diabetes (blood glucose ≥ 250 mg/dl) and severe diabetes (blood glucose ≥ 400 mg/dl) starting at week 14 of the study. However, during the later stages of the study (i.e., after week 23), the 2-mg/kg dose had no effect on disease incidence. In animals treated with 6-mg/kg and 20-mg/kg GEN, significant decreases in the total incidence of diabetes were observed starting at week 16, while the incidence of severe diabetes was significantly decreased with the changes being observed initially at weeks 18 and 17 for the 6-mg/kg and 20-mg/kg GEN treatment groups, respectively. Several lines of evidence, including histopathological analysis, suggested that GEN protected the pancreas from autoimmune destruction. However, this protective effect of GEN was absent when female NOD mice were maintained on NTP-2000 rodent diet, which contained 5% soybean meal and 7.5% alfalfa meal (the total concentrations of phytoestrogens ranged between 95 and 134 mg/kg). In summary, oral dosing of GEN reduced the incidence and increased the time to onset of T1D in female NOD mice but only when fed a soy- and alfalfa-free diet.

Thalidomide modulation of the immune response in female B6C3F1 mice: a host resistance study

Previously, we have reported that thalidomide (Thd) treatment can modulate the immune responses in female B6C3F1 mice. The present study was designed to evaluate whether or not these immunomodulatory responses were of sufficient magnitude to alter host resistances in a number of pathogen and tumor models. B6C3F1 mice were treated intraperitoneally with Thd (30-150 mg/kg) for 14 or 28 days, then inoculated with either Plasmodium yeolii, PYB6 fibrosarcoma tumor cells, B16F10 melanoma tumor cells, Listeria monocytogenes, or Streptococcus pneumoniae. Significant dose-dependent protection against B16F10 and L. monocytogenes was observed in mice that were treated with Thd. Furthermore, time course study using bacterial colony-forming units per spleen and liver as the endpoints indicated that the protective effect of Thd on host resistance to L. monocytogenes was time-dependent. In contrast, Thd treatment did not affect host resistance to P. yeolii, S. pneumoniae and PYB6 tumor. Additionally, the effect of Thd on the phagocytic function of the mononuclear phagocyte system (MPS) was evaluated following intravenous injection of 51Cr-labeled sRBCs. The overall phagocytic activity of MPS was not significantly altered by Thd treatment. In conclusion, these results demonstrate that Thd immunomodulation altered host resistance to B16F10 and L. monocytogenes; and selective modulation of Thd on the immune system may be responsible for the pathogen or tumor-specific effect of this compound.

Immunotoxicological profile of chloroform in female B6C3F1 mice when administered in drinking water

Chloroform can be formed as a disinfection by-product during water chlorination, one of the primary modalities for purifying municipal water supplies for human consumption. The aim of this study was to characterize the immunotoxic effects of chloroform in female B6C3F1 mice when exposure occurred via the drinking water. Consistent with human exposure, female B6C3F1 mice were exposed to chloroform-containing drinking water at 2.5, 10, 25, 100, and 250 ppm for 28 days. The examined endpoints included the effects of chloroform on body and organ weights, water consumption, hematology, innate immunity, humoral immunity, and cell-mediated immunity. The functions of natural killer, B-, and T-cells were not altered by chloroform in drinking water at the concentrations tested, except that an increase in splenocyte basal proliferation was observed at chloroform levels of 100 and 250 ppm. Following chloroform administration, there was a decreased number of circulating neutrophils in the blood in all treatment groups, but neutrophil function in lung homogenates, as evaluated using an assay for myeloperoxidase activity following lipopolysaccharide and N-Formyl-Met-Leu-Phe stimulation, was not compromised. Further, the results of host resistance to Listeria monocytogenes infection also suggested that neutrophil function was normal. At the highest treatment level of chloroform (250 ppm), erythrocyte number and hemoglobin levels were significantly decreased. Some significant changes were also observed for body weights, water consumption, and organ weights; however, most of these effects were only observed at the highest treatment level of chloroform (250 ppm). Taken together, the results demonstrate that while chloroform administered via the drinking water affects body weight and selected hematological parameters at high dose levels, overall immune responses, as measured in several tests for immune function, are not compromised.

Carbon tetrachloride is immunosuppressive and decreases host resistance to Listeria monocytogenes and Streptococcus pneumoniae in female B6C3F1 mice

Carbon tetrachloride (CCl(4)) is an environmental contaminant that has been detected in ambient air, seawater, surface-water and snow. The immunotoxic potential of CCl(4) was evaluated in female B6C3F1 mice. The animals were administered with CCl(4) daily for 14 days at doses of 50, 100, 500 or 1000 mg/kg body weight by gavage with corn oil as a vehicle. Exposure to CCl(4) resulted in an increase of liver weight but not the body weight and the weights of brain, spleen, lungs, thymus and kidneys. Exposure to CCl(4) produced minimal effect on differential hematological parameters; however, it produced a significant increase in serum glutamic-pyruvic transaminase (SGPT) levels in all dose groups while other serum chemistries showed sporadic increases, primarily at the dose level of 1000 mg/kg. Exposure to CCl(4) produced a decreased humoral immune response; the IgM antibody forming cell (AFC) response to sheep red blood cells (sRBC) was suppressed with the maximal decrease (45%) observed at the dose level of 1000 mg/kg. The IgM serum titer to sRBC was also reduced with a maximal decrease (54%) observed at the dose level of 500 mg/kg. Although exposure to CCl(4) had no effects on the mixed leukocyte response (MLR), cytotoxic T lymphocyte activity and natural killer (NK) cell activity, a decrease in both the absolute number and the percentage of CD4(+)CD8(-) at the dose level of 500 mg/kg was observed. The functional activity of the mononuclear phagocyte system was compromised as reflected by a decrease in the vascular clearance of (51)Cr-sRBC and a decrease in the uptake of (51)Cr-sRBC by the liver. Finally, in the two host resistance models evaluated, exposure to CCl(4) decreased host resistance to both Streptococcus pneumoniae and Listeria monocytogenes with greater susceptibility to the latter. Overall, these studies demonstrate that CCl(4) was immunosuppressive in female B6C3F1 mice.

Differential STAT5 activation and phenotypic marker expression by immune cells following low levels of ethanol consumption in mice.

ABSTRACT Ethanol has been recognized as an immunosuppressive agent for many years. Effects of high levels of ethanol consumption on immune functions have been extensively studied, but little is known about the effects of low levels (scuh as 5% ethanol) of ethanol consumption. Herein we report that exposure of mice to 5% ethanol for 4–8 weeks decreases IL-2-augmented splenic NK cell activity, decreases the numbers of NK cells in spleen and liver, decreases the number of granulocytes (Gr-1+) in bone marrow and spleen, and decreases the percentages of B cells in liver. In contrast, the percentages of CD4+CD8+ thymocytes, CD4+CD8− splenocytes, CD4+CD8− liver nonparenchymal cells, CD3+ splenocytes, and CD3+ bone marrow cells were increased. Furthermore, exposure to 5% ethanol increases STAT5 activation in T cells and liver cells while decreases STAT5 activation in NK cells. Taken together, these findings suggest that low levels of ethanol consumption can differentially modulate immune cells in thymus, spleen, bone marrow and liver, which may be due to differential regulation of STAT5 activation by ethanol.

Immunotoxicity of sodium bromate in female B6C3F1 mice : A 28-day drinking water study

Bromate is one of the water disinfection by-products (DBPs) produced during the process of ozonation. The purpose of this study was to evaluate the immunotoxic potential of sodium bromate (SB) in female B6C3F1 mice. SB was administered in the drinking water for 28 days at doses of 80–800 mg/l. There was no difference in drinking water consumption between the animals exposed to SB and the tap water controls. Exposure to SB did not produce any signs of overt toxicity. Furthermore, no significant differences were observed in body weight, body weight gain, or the weights of thymus, liver, kidneys or lungs. No gross pathological lesions were observed in SB-treated animals. However, animals exposed to SB had a significant increase in absolute (28%) and relative (26%) spleen weights. The erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), platelet count, total leukocyte count, and counts of differential leukocytes were unaffected by SB. A dose-related increase in reticulocytes was observed following exposure to SB with the greatest increase (78%) observed at the highest dose level. Overall, there were no changes in the absolute number of total T cells, CD4+CD8− T cells, CD4−CD8+ T cells, natural killer (NK) cells and macrophages. Exposure to SB did not affect the percentage of B cells, although a slight increase in absolute number of B cells at the dose of 600 mg/l was observed. There was no alteration in IgM antibody-forming cell (AFC) response, mixed leukocyte reaction (MLR) and NK cell activity after exposure to SB. When the activity of peritoneal macrophages, unstimulated or stimulated with IFN-γ and LPS, was evaluated using the cytotoxic/cytostatic assay of B16F10 tumor cells, the suppressive effect of macrophages on the proliferation of B16F10 tumor cells was decreased after exposure to SB. In conclusion, SB, when administered in the drinking water at doses from 80 mg/l to 800 mg/l, produced minimal toxicological and immunotoxic effects in female B6C3F1 mice.

Immunotoxicity of dibromoacetic acid administered via drinking water to female B6C3F1 mice

Dibromoacetic acid (DBA) is a disinfection by-product commonly found in drinking water as a result of chlorination/ ozonation processes. The Environmental Protection Agency estimates that more than 200 million people consume disinfected water in the United States. This study was conducted to evaluate the potential immunotoxicological effects of DBA exposure when administered for 28 days via drinking water to B6C3F1 mice, at concentrations of 125, 500, and 1000 mg/L. Multiple endpoints were evaluated to assess innate, humoral, and cell-mediated immune components, as well as host resistance. Standard toxicological parameters were unaffected, with the exception of a dose–responsive increase in liver weight and a decrease in thymus weight at the two highest exposure levels. Splenocyte differentials were affected, although the effects were not dose–responsive. Exposure to DBA did not significantly affect humoral immunity (immunoglobulin M [IgM] plaque assay and serum IgM anti-sheep erythrocyte titers) or cell-mediated immunity (mixed-leukocyte response). No effects were observed on innate immune function in either interferon-γ-induced in vitro macrophage cytotoxic activity or basal natural killer (NK)-cell activity. Augmented NK-cell activity (following exposure to polyinosinic-polycytidylic acid) was decreased at the low dose, however the effect was not dose–responsive. Finally, DBA exposure had no effect on resistance to infection with either Streptococcus pneumoniae or Plasmodium yoelii, or challenge with B16F10 melanoma cells. With the exception of changes in thymus weight, these results indicate that DBA exposure resulted in no immunotoxic effects at concentrations much larger than those considered acceptable in human drinking water.

Contact sensitizing potential of pyrogallol and 5-amino-o-cresol in female BALB/c mice.

Hair dye components such as pyrogallol and cresol have been shown previously to promote allergic reactions such as rashes, dermal inflammation, irritation and dermatitis. The objective of this study was to determine the contact sensitization potential of pyrogallol (PYR) and 5-amino-o-cresol (AOC) when applied dermally to female BALB/c mice. Measurement of the contact hypersensitivity response was initially accomplished using the local lymph node assay. For PYR, significant increases in the proliferation of lymph node cells were observed at concentrations of 0.5% (w/v) and higher. For AOC, borderline increases, albeit significant, in auricular lymph node cell proliferation were observed at 5% and 10%. Results from the irritancy assay suggested that PYR, but not AOC, was an irritant. To further delineate whether PYR was primarily an irritant or a contact sensitizer, the mouse ear swelling test (MEST) was conducted. A significant increase in mouse ear thickness was observed at 72h following challenge with 0.5% PYR in mice that had been sensitized with 5% PYR. In contrast, no effects were observed in the MEST in mice sensitized and challenged with the highest achievable concentration of AOC (10%). Additional studies examining lymph node subpopulations and CD86 (B7.2) expression by B cells further support the indication that PYR was a sensitizer in BALB/c mice. The results demonstrate that PYR is both a sensitizer and an irritant in female BALB/c mice. However, the contact sensitization potential of AOC is minimal in this strain of mouse.