Suppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the high-affinity antibody response in C57BL/6 mice.

In the humoral immune response to an invasion of foreign antigens, B cells differentiate into low-affinity antibody-forming cells (AFCs) that mainly secrete IgM or, through germinal center (GC) formation, into high-affinity AFCs that secrete IgG-class antibodies with a higher affinity for the antigen. Previous studies have established the suppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on low-affinity antibody responses to antigens. However, whether and how TCDD affects the high-affinity antibody response to antigens has not yet been clarified. In this paper we investigate the effects of TCDD on GC formation, high-affinity AFC generation, and high-affinity antibody production in the primary humoral immune response. C57BL/6 mice were orally administered 0 or 20 microg/kg of TCDD and subsequently immunized with alum-precipitated ovalbumin (OVA) on day 0. Then the GC formation in the spleen and OVA-specific antibodies in the plasma, was evaluated until day 14 postimmunization. TCDD exposure reduced the production of OVA-specific IgG1 on days 10 and 14. GC formation in the spleen was also suppressed by TCDD exposure, and the suppression persisted from day 7 until day 14. In TCDD-administered mice, on day 7, cellular proliferation in the GCs was significantly suppressed, although apoptosis was not markedly affected. In order to measure high-affinity antibody and high-affinity AFCs, the mice were administered TCDD followed by immunization with alum-precipitated (4-hydroxy-3-nitrophenyl) acetyl linked to chicken gamma-globulin (NP-CG). The frequency of high-affinity NP-specific AFCs that bind to low-haptenated antigen was clearly shown to be reduced in the spleen on days 10 and 14. Furthermore, the high-affinity anti-NP IgG1 levels on days 10 and 14 postimmunization were significantly reduced by TCDD exposure. Taken together, the results of this paper demonstrate that TCDD exposure inhibits the generation of high-affinity AFCs and high-affinity antibody production during the primary humoral immune response and suggest that these alterations were caused by the suppression of antigen-responding B-cell proliferation induced by TCDD during GC formation.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cell-derived cytokine production in ovalbumin (OVA)-immunized C57Bl/6 mice

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress both cellular and humoral immunity. Effector T cell-derived type-2 cytokines, including IL-4 and IL-5, play pivotal roles in humoral immunity. Herein, we studied whether TCDD affects type-2 cytokine productions during the immune response. C57Bl/6 mice were intraperitoneally immunized with ovalbumin (OVA) and orally administered 5 or 20 microg TCDD/kg on Day 0, and then challenged with OVA on Day 21. Seven days later (Day 28), antigen-specific antibodies in plasma, and T cell-derived cytokines produced by splenocytes and proliferation of splenocytes upon ex vivo re-stimulation with OVA were investigated. The quantities of IgM class and IgG1 class OVA-specific antibodies in plasma were reduced by 5 or 20 microg TCDD/kg and by 20 microg TCDD/kg, respectively. While thymus weight and cellularity were reduced by 20 microg TCDD/kg, spleen weight and cellularity were not changed by either 5 or 20 microg TCDD/kg. The proportions of B and T cells in the spleen were not affected by TCDD exposure. On the other hand, splenocytes from mice treated with 5 or 20 microg TCDD/kg were shown to produce less IL-4 or IL-5 upon ex vivo re-stimulation with OVA. Production of the T cell growth factor IL-2 was also decreased in splenocytes from TCDD-treated mice. In contrast, the type-1 cytokine IFN-gamma was increased by TCDD. Twenty micrograms of TCDD/kg suppressed OVA- or T cell mitogen (Con A)-stimulated proliferation of splenocytes, but did not affect B cell mitogen (LPS)-stimulated proliferation. These results suggested compromised T cell activation and suppressed type-2 cytokine production by T cells to be involved in the impaired humoral immunity associated with TCDD exposure.     

Modulation of serum complement levels following exposure to polychlorinated dibenzo-p-dioxins

Subchronic 14-day exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed serum total hemolytic complement activity (CH50) in female B6C3F1 mice at doses of 0.01, 0.05, 0.1, 0.5, 1.0, and 2.0 micrograms/kg. Serum levels of complement component C3 were also suppressed at doses of 0.5, 1.0, and 2.0 micrograms/kg. Another dioxin isomer, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HCDD), also produced dose-dependent suppression of complement activity at doses of 0.1, 1.0, and 10 micrograms/kg with decreased C3 levels at 10 micrograms/kg. Both TCDD and HCDD enhanced susceptibility to Streptococcus pneumoniae, a bacterial pathogen whose host defense is complement mediated. Recovery studies demonstrated that complement activity in TCDD (1 microgram/kg) and HCDD (10 micrograms/kg)-treated animals was suppressed until 50 days post-treatment, while low doses of HCDD (0.1 and 1.0 micrograms/kg) elevated CH50 levels. Acute exposure to TCDD (14 micrograms/kg) also suppressed complement CH50 and C3 levels. These studies demonstrate that the complement system and innate immunity represent potential target sites for polychlorinated dibenzo-p-dioxins.     

Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known to man. Hassoun et al. (1998, Toxicol. Sci. 42, 23-27) reported an increase in the production of reactive oxygen species (ROS) in the brain of female B6C3F1 mice following subchronic exposure to TCDD at doses as low as 0.45 ng/kg/day. In the present study, oxidative stress was characterized in liver, spleen, lung, and kidney following subchronic (0.15-150 ng/kg; 5 days/week for 13 weeks, po) or acute exposure (0.001-100 microg/kg, po) to TCDD in order to investigate the interaction between tissue concentration and time for production of ROS. Seven days following acute administration of TCDD, mice were sacrificed; they demonstrated increases in liver superoxide anion production (SOAP) and thiobarbituric acid reactive substances (TBARS) at doses of 10 and 100 microg/kg, associated with hepatic TCDD concentrations of 55 and 321 ng/g, respectively. Liver obtained from mice following subchronic TCDD exposure demonstrated an increase in SOAP and TBARS above controls at doses of 150 ng/kg/day with liver TCDD concentration of only 12 ng/g. Interestingly, glutathione (GSH) levels in lung and kidney following sub-chronic TCDD exposure were decreased at the low dose of 0.15 ng/kg/day. This effect disappeared at higher TCDD doses. The data suggest that higher tissue TCDD concentrations are required to elicit oxidative stress following acute dosing than with subchronic TCDD exposure. Therefore, the mechanism of ROS production following TCDD exposure does not appear to be solely dependent upon the concentration of TCDD within the tissue. In addition, very low doses of TCDD that result in tissue concentrations similar to the background levels found in the human population produced an effect on an oxidative stress endogenous defense system. The role of this effect in TCDD-mediated toxicity is not known and warrants further investigation.     

Protective effects of vitamin A and vitamin E succinate against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced body wasting, hepatomegaly, thymic atrophy, production of reactive oxygen species and DNA damage in C57BL/6J mice

The protective effect of vitamin A and vitamin E succinate against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced acute toxicity and measures of oxidative stress was studied. Ten mice were treated with either vitamin A (50 mg/kg every other day for eight days) or vitamin E succiante (150 mg/kg/day followed by a dose of 40 mg/kg/day for five additional days). Half of each of the above groups of animals received TCDD on day 4. Five mice received corn oil or TCDD alone. After five days of TCDD treatment, antioxidant combination treatment with vitamin A and TCDD or vitamin E succinate and TCDD resulted in a significant reduction in indicators of acute toxicity including the decrease in total body and thymus weight as compared to TCDD alone (P<0.05). The combination treatment produced also a significant reduction in the increase in liver weight as compared to TCDD only (P<0.05). Following one day of treatment with 50 microg TCDD/kg, vitamin A and vitamin E succinate produced a significant decrease in the production of superoxide anion by peritoneal lavage cells (P<0.05) and in DNA-single strand breaks in the same cells (P<0.05) as assessed by the reduction of cytochrome c and the alkaline elution technique, respectively. A significant decrease in DNA-single strand breaks in peritoneal lavage cells was observed following 5 days treatment with 50 microg TCDD/kg (P<0.05). The results indicate a potential role for oxidative stress in the acute toxicity of TCDD and a protective effect for vitamin A and vitamin E succinate in the overall toxicity of TCDD including measures of oxidative stress.     

Protective effects of tea melanin against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity: antioxidant activity and aryl hydrocarbon receptor suppressive effect

We examined the protective ability of tea melanin against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity in C57BL6J mice. Reduced tea melanin (RTM) and non-reduced tea melanin (NRTM) were incorporated to distinguish anti-oxidant activity from alternative pathways. The mice were given a single oral dose of TCDD (100 microg/kg body weight) and then they were administered daily with NRTM or RTM (40 mg/kg, p.o.) for next 14 d. RTM protected the animals against TCDD-induced lipid peroxidation, inhibition of glutathione peroxidase, alteration in reduced and oxidized glutathione concentrations, loss of body weight, and increased relative liver weight. NRTM was less effective as compared to RTM because of its inferior antioxidant activity, but it still displayed a strong protective effect against TCDD toxicity owing to its similar suppression of the activity of the aryl hydrocarbon receptor. Both NRTM and RTM suppressed the expression of CYP1A1 gene and prevented the activation of cytochrome P450 isozyme in the livers of animals exposed to TCDD. These results suggest that tea melanin might be a potential agent offering dual protection against the development of TCDD-induced oxidative stress.     

Melatonin attenuates lipopolysaccharide (LPS)-induced apoptotic liver damage in D-galactosamine-sensitized mice

D-Galactosamine (GalN) depletes UTP primarily in liver, resulting in decreased RNA synthesis in hepatocytes. When given together with a sublethal dose of lipopolysaccharide (LPS), GalN highly sensitizes animals to produce apoptotic liver injury with severe hepatic congestion, resulting in rapid death. Melatonin is a cytokine modulator, antioxidant and anti-apoptotic agent. In the present study, we investigated the effect of melatonin on LPS-induced apoptotic liver damage in GalN-sensitized mice. Female CD-1 mice were intraperitoneally (i.p.) injected with melatonin (5.0mg/kg) 30min before GalN/LPS (700mg10microg/kg, i.p.), another two doses of melatonin (2.5mg/kg, i.p.) being administered 1 and 2h after GalN/LPS. Results showed that serum alanine aminotransferase (ALT) activities were markedly increased 8h after GalN/LPS treatment, massive hemorrhage being observed in histological sections of liver from GalN/LPS-treated mice. Melatonin significantly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, melatonin distinctly improved GalN/LPS-induced congestion. Additional experiment showed that melatonin significantly attenuated GalN/LPS-induced hepatic apoptosis, measured by inhibition of caspase-3 activities and attenuation of DNA laddering. Furthermore, melatonin markedly increased hepatic Se-dependent glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) activities and attenuated hepatic glutathione (GSH) depletion in GalN/LPS-treated mice. Increases in serum tumor necrosis factor alpha (TNF-alpha), which were observed in GalN/LPS-treated mice, were significantly reduced by melatonin. However, melatonin had no effect on LPS-evoked nitric oxide production in GalN-sensitized mice. Taken together, these results indicate that melatonin protected against LPS-induced liver damage in GalN-sensitized mice through its strong ROS-scavenging, antiinflammatory and antiapoptotic effects.     

Persistent suppression of delayed-type hypersensitivity in adult F344 rats after perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Recently we observed a suppressed delayed-type hypersensitivity (DTH) response to bovine serum albumin (BSA) in the 4-5-month-old offspring of F344 rat dams receiving as little as 1.0 microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg on gestational day (GD) 14. This study was designed to characterize better this suppression of the DTH response. First, the persistence of the DTH suppression was determined by measuring the DTH response to BSA in the offspring of dams dosed orally with 3.0 microg TCDD/kg on GD14 as well as in age-matched controls at 4, 8, 12 and 19 months of age. TCDD significantly suppressed the males' DTH response through 19 months of age. While the females' DTH response was reduced at 8, 12 and 19 months, significant suppression was observed only at 4 months of age. Secondly, the lowest maternal dose of TCDD that produced DTH suppression was determined by measuring the DTH response to BSA in the 4- and 14-month-old offspring of dams dosed orally with 0, 0.1, 0.3 or 1.0 microg TCDD/kg on GD14. In the males, suppression was observed at a maternal dose as low as 0.1 microg TCDD/kg at 14 months of age, while a maternal dose of 0.3 microg TCDD/kg was necessary to cause suppression in the 14-month-old females. Both males and females were more sensitive to the suppression at 14 months of age than at 4 months of age. Lastly, the DTH response to a second antigen was examined by measuring the DTH response to either BSA or keyhole limpet hemocyanin (KLH) in the 5- or 4-month-old male offspring, respectively, of dams dosed orally with either 0 or 3.0 microg TCDD/kg on GD14. The DTH response to both antigens was suppressed significantly. Phenotypic analysis was performed on thymus and lymph node suspensions. Significant effects in the thymus included an increased percentage of gammadelta TCR+ cells and a decreased percentage of gammadelta TCR+/CD4- CD8- and MHCI- MHCII- cells. In the popliteal lymph node draining the BSA-injected footpad, there was a decreased percentage of gammadelta TCR+ and MHCI- MHCII- cells and an increased percentage of MHCI+ cells. In conclusion, the suppression of the DTH response associated with perinatal TCDD exposure is persistent through late adulthood, occurs at a low dose (i.e. 0.1 microg TCDD/kg) to the dam, and is more pronounced in males than females. While phenotypic analysis identified differences in subsets of thymocytes and lymph node cells between control and TCDD exposed offspring, no clear correlations were established between altered subpopulations and suppressed DTH responses.     

Pharmacokinetics and biological activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver and adipose tissue, and hepatic ethoxyresorufin O-deethylase (EROD) activity were studied subsequent to a single subcutaneous injection of TCDD. Two types of experiments were performed to study: (a) time-dependent changes following a single injection of 300 ng TCDD/kg body wt (points 1-4), and (b) dose-dependent changes measurable after 7 days following a single injection (points 5-7). 1. Absorption of TCDD following a single subcutaneous injection was about 90% after 3 days and 98% after 5 days. 2. Following a single dose of 300 ng TCDD/kg body wt peak concentrations were: liver (after 3 days): 4.7 +/- 0.9 ng/g wet wt, and adipose tissue (after 7 days): 0.82 +/- 0.07 ng/g wet wt. 3. T1/2 of TCDD in liver was 13.6 days over the total experimental period (from day 10 to 91 of the study), apparently with an initial faster phase: 11.5 days (from day 10 to 49), and a slower period at the end of the experiment: 16.9 days (from day 49 to 91); in adipose tissue the t1/2 was 24.5 days (from day 14 to 91 of the study). 4. Maximum induction of EROD in the liver was observed (14-fold at 300 ng TCDD/kg body wt) 3-7 days following the injection; the activity was decreased to about one third of the maximum 3 weeks after the injection; increase in total cytochrome P-450 at this dose was only about 1.4-fold at the induction maximum. 5. The ratio of the TCDD concentrations in liver and adipose tissue increased considerably between doses of 3 ng TCDD/kg body wt (ratio: about 0.74) and 3000 ng TCDD/kg body wt (ratio: about 7.7). 6. The extent of EROD induction in the liver increased dose dependently. A significant effect was first observed with a dose of 3 ng TCDD/kg body wt (activity about +32% above control activity). The corresponding tissue concentration was about 10 pg TCDD/g liver wet wt. 7. An almost perfect linear relationship exists (when using a double-log plot) between the hepatic TCDD concentration and the EROD activity for tissue concentrations ranging from 40 to 30,000 pg TCDD/g wet wt.     

Deterioration of spatial learning performances in lipopolysaccharide-treated mice

It is well demonstrated that acute or chronic stress leads to reduction of learning ability. Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, induces profound physiological and behavioral changes, including fever, decrease in food motivation, and decrease in social behavior. These changes might be interpreted as an acute stress reaction to the LPS. In the present study, therefore, we investigated the effects of LPS (400-800 microg/kg, i.p.) on spatial learning performances using C57BL/6J male mice. In the Morris water-maze task, spatial learning performances were examined in six trials of training for two consecutive days. LPS-treated mice took a longer time to reach the hidden platform than control mice (F(1,60)=4.80801, P<0.05 at 600 microg/kg). In addition, injection of LPS decreased the percent of correct choices in the Y-maze test (P<0.05 at 800 microg/kg). LPS, however, did not alter the body weight, grip tone, motor activity or swimming speed. Taken together, these results indicate that LPS treatment specifically impaired spatial learning performances.     

Effects of bacterial lipopolysaccharide on phenobarbital-induced CYP2B expression in mice.

Models of inflammation and infection, such as bacterial lipopolysaccharide (LPS), cause suppression of cytochrome P450 expression in various species, although the mechanisms involved are poorly understood. The effects of LPS on expression of phenobarbital (PB)-induced CYP2B1/2 in rats have been well characterized, but less is known about the effects of LPS on PB-induced CYP2B in mice. Since genetically manipulated mice represent an attractive model to study the mechanisms involved in the down-regulation of CYP2B expression by LPS, we investigated the effects of LPS on PB-induced CYP2B expression in mouse liver. Female C57BL/6 mice were injected with 100 mg/kg PB once daily for 4 days to induce CYP2B10 expression, and 1 mg/kg LPS was injected i.p. with the last PB dose. LPS inhibited the mRNA expression of CYP2B10 and CYP2B9 at 6 and 12 h of treatment, with the inhibitory effect more profound at 12 h. LPS also suppressed the CYP2B9 mRNA level at 24 h. However, CYP2B10 mRNA levels in mice treated with PB alone had declined markedly by 24 h after the last PB injection; therefore, no effect of LPS could be discerned. Further experiments showed that injections of 33 mg/kg PB every 8 h produced more stable CYP2B10 mRNA and enzymatic activity. Suppression of CYP2B protein level was found in LPS-treated animals at 24 h of treatment, although no significant effects were noticed at 6 and 12 h of treatment. This study suggests that LPS suppresses the expression of phenobarbital-induced CYP2B expression in mice, which resembles its effects in rats.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) alter body weight by decreasing insulin-like growth factor I (IGF-I) signaling.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects glycemia due to reduced gluconeogenesis; when combined with a reduction in feed intake, this culminates in decreased body weight. We investigated the effects of steady-state levels of TCDD (loading dose rates of 0.0125, 0.05, 0.2, 0.8, and 3.2 microg/kg) or approximately isoeffective dose rates of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) (loading dose rates of 0.3125, 1.25, 5, 20, and 80 microg/kg) on body weight, phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression and activity, and circulating concentrations of insulin, glucose, and insulin-like growth factor-I (IGF-I), and expression of hepatic phosphorylated AMP kinase-alpha (p-AMPK) protein in female Sprague-Dawley rats (approximately 250 gm) at 2, 4, 8, 16, 32, 64, and 128 days after commencement of treatment. At the 0.05 and 1.25 microg/kg loading dose rates of TCDD and HxCDD, respectively, there was a slight increase in body weight as compared to controls, whereas at the 3.2 and 80 microg/kg loading dose rates of TCDD and HxCDD, respectively, body weight of the rats was significantly decreased. TCDD and HxCDD also inhibited PEPCK activity in a dose-dependent fashion, as demonstrated by reductions in PEPCK mRNA and protein. Serum IGF-I levels of rats treated initially with 3.2 microg/kg TCDD or 80 microg/kg HxCDD started to decline at day 4 and decreased to about 40% of levels seen in controls after day 16, remaining low for the duration of the study. Eight days after initial dosing, hepatic p-AMPK protein was increased in a dose-dependent manner with higher doses of TCDD and HxCDD. There was no effect with any dose of TCDD or HxCDD on circulating insulin or glucose levels. In conclusion, doses of TCDD or HxCDD that began to inhibit body weight in female rats also started to inhibit PEPCK, inhibited IGF-I, while at the same time inducing p-AMPK.     

Effect of a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin on immune function in male NC/Nga mice.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immunosuppression in humans and animals. However, the effect of TCDD on Th2-type immune responses such as allergic reactions has been unclear. Using NC/Nga mice that developed atopic dermatitis-like skin lesions with marked elevation in plasma of total IgE when bred under conventional conditions, we investigated the effects of a single oral dose of TCDD on immune responses. NC/Nga mice received a single oral dose (0 or 20 microg/kg body weight) of TCDD. On day 7, treatment with TCDD alone decreased the cellularity of thymus. However, treatment with TCDD modified the cellularity of spleens and mesenteric lymph nodes (MLNs) but not of the thymus on day 28. When NC/Nga mice received ip immunization with OVA and alum on the same day as the TCDD treatment (0, 5, or 20 microg/kg body weight), TCDD markedly suppressed the concentrations of Th2-type cytokines (e.g., IL-4 and IL-5) in culture supernatants of spleen cells, whereas IFN-gamma production significantly increased. TCDD exposure reduced anti-OVA and total IgE antibody titers in plasma and did not induce the development of atopic dermatitis-like lesions in the pinnae or dorsal skin of NC/Nga mice. These results suggest that in NC/Nga mice, exposure to TCDD may impair the induction of Th2-type immune responses.     

Effects of aging on resistance to Trichinella spiralis infection in rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Immune function, including resistance to infection, decreases as humans and rodents age. We have shown that preinfection exposure of young (9-11 weeks) mice or rats to TCDD decreased resistance to Trichinella spiralis (Ts) infection, expressed as delayed onset or completion of parasite elimination and as increased muscle burdens of larvae. It has also been shown that aged mice express lower constitutive levels of resistance to Ts infection, compared to young adult animals. This study tested the hypothesis that the age-related decrease in constitutive levels of resistance to Ts infection exacerbates the decreased resistance to infection that follows TCDD exposure. This hypothesis addresses the concern that TCDD may pose a greater threat to the elderly than to the population at large. Animals were given a single oral dose of 1, 10, or 30 microg TCDD/kg, 7 days before infection. Eleven days later, young (approximately 10 weeks) control rodents had eliminated a greater proportion of the original parasite burden from the intestine than aged control animals. Nevertheless, parasite elimination was decreased by TCDD exposure only in young rodents. The effect of TCDD exposure on numbers of encysted larvae was evaluated only in rats. Increased larvae burdens occurred in young rats at 30 microg TCDD/kg and at 10 or 30 microg TCDD/kg in aged rats. Parasite-specific splenocyte and lymph node cell proliferation was suppressed following dioxin exposure in young mice; cells from aged mice were markedly less responsive to stimulation, yet less sensitive to TCDD exposure. The response to parasite antigens was not affected in aged rats exposed to TCDD, although elevated mitogen-driven B-cell proliferation was observed. These results indicate that age-related constitutive immunosuppression did not exacerbate TCDD-induced suppression of T-cell mediated adult parasite expulsion; rather, advanced age provided some degree of protection. On the other hand, a lower dose of TCDD was required in aged rats to suppress the combined humoral and cellular responses that limit the burden of encysted larvae, compared to young rats. These model-dependent results preclude acceptance or rejection of the tested hypothesis.     

Persistent, low-dose 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: effect on aryl hydrocarbon receptor expression in a dioxin-resistance model

Most toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the aryl hydrocarbon receptor (AHR). A single, acute dose of TCDD can alter its own receptor levels thus complicating evaluation of dose-response relationships for AHR-mediated events. Since environmental exposure to dioxins is typically of a repeated low-dose nature, we examined the effect of such exposure on AHR expression. Three rat strains differing greatly in their sensitivity to acute TCDD lethality, Long-Evans (Turku AB) (L-E) (LD50 approximately 10 microg/kg); Sprague Dawley (SD) (LD50 approximately 50 microg/kg); and Han/Wistar (Kuopio) (H/W) (LD50 > 9600 microg/kg), were administered TCDD intragastrically, biweekly for 22 weeks producing doses equivalent to 0, 10, 30, and 100 ng/kg/day. Changes in hepatic AHR levels were quantitated at the protein level by radioligand binding and immunoblotting and at the mRNA level by RT-PCR. Cytosolic AHR protein was elevated at 10 or 30 ng/kg/day TCDD in SD and L-E rats; AHR mRNA was also elevated at these doses, suggesting a pretranslational mechanism. There was no apparent relationship between TCDD-induced AHR regulation and strain sensitivity to TCDD. Overall, "subchronic" TCDD did not greatly perturb AHR expression. The maintenance of relatively constant receptor levels in the face of persistent agonist stimulation is in contrast to the sustained depletion of AHR by TCDD observed in cell culture and to the fluctuations in AHR observed hours to days following acute TCDD exposure in vivo. Changes in AHR levels may affect dose-response relationships; the effect of TCDD on its own receptor at environmentally relevant dosing schemes is therefore important to risk assessment.     

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin administration and high-fat diet on the body weight and hepatic estrogen metabolism in female C3H/HeN mice

We studied the effect of administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by i.p. injection once every 2 weeks in combination with a high-fat (HF) diet for 8 or 16 weeks on the body and organ weight changes as well as on the hepatic enzyme activity for estrogen metabolism in C3H/HeN female mice. Administration of TCDD at 100 microg/kg b.w. once every 2 weeks for 8 weeks increased the body weight by 46% in the HF diet-fed animals, but not in the regular diet-fed animals. This is the first observation suggesting that TCDD at a high dose (100 microg/kg b.w.), but not at lower doses (1 or 10 microg/kg b.w.), may have a strong obesity-inducing effect in C3H/HeN mice fed an HF diet. While TCDD increased liver weight and decreased thymus weight in animals, these effects were enhanced by feeding animals an HF diet. Metabolism studies showed that TCDD administration for 8 or 16 weeks increased the liver microsomal activity for the 2- and 4-hydroxylation of 17 beta-estradiol in animals fed a control diet, but surprisingly not in animals fed an HF diet. Treatment with TCDD dose-dependently increased the hepatic activity for the O-methylation of catechol estrogens in both control and HF diet-fed animals, and it also decreased the levels of liver microsomal sulfatase activity for hydrolysis of estrone-3-sulfate. TCDD did not significantly affect the hepatic enzyme activity for the glucuronidation or esterification of endogenous estrogens. It is suggested that enhanced metabolic inactivation of endogenous estrogens by hepatic estrogen-metabolizing enzymes in TCDD-treated, control diet-fed animals contributes importantly to the reduced incidence of estrogen-associated tumors in animals treated with TCDD.     

Fetal thymus organ culture as an in vitro model for the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin and its congeners

Fetal thymuses from C57BL/6 (B6) and DBA/2J (D2) mice from gestation day 14 or 15 were explanted and grown for 2 and 6 days in culture in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and a number of its congeners, known ligands of the Ah receptor (Ah, designating genetic locus for aryl hydrocarbon responsiveness). TCDD and 2,3,7,8-tetrachlorodibenzofuran (TCDBF) showed the same toxicity to B6 thymuses with a 50% inhibition of lymphoid development (EC50) at 10(-10) M concentration. 3,3',4,4'-Tetrachloroazoxybenzene (TCAOB) was only 2-10 times less effective, while the EC50 of 3,3',4,4'-tetrachlorobiphenyl (TCB) was around 10(-8) M (100 times higher than that of TCDD). TCBs with chlorine atoms in the position close to the biphenyl bridge were nontoxic even at 10(-5) M concentration. Thymuses exposed to TCDD, TCDBF, and TCAOB in vivo at teratogenic doses given to the mothers and explanted 24-48 hr later were smaller and inhibited in their early in vitro growth, but recovered slowly (less rapid for TCDD) as judged by lymphoid cell counts and [3H]thymidine incorporation. These results indicate a good correlation for this group of compounds between their activity as ligands of the Ah receptor and toxicity in vitro. Other ligands of the Ah receptor, namely 3-methylcholanthrene and beta-naphthoflavone, were inactive at the highest concentrations tested (10(-6) M). Thymuses from D2 mice, considered Ah receptor-defective, were nonsensitive to TCDD at the concentrations used (up to 3 X 10(-8) M) after 2 days in culture, indicating more than 100 times lower sensitivity as compared to B6 thymuses. After 6 days in culture, their sensitivity was however only 1 order of magnitude lower than that of B6 thymuses. Therefore "low sensitivity" of D2 thymuses may be at least partially overcome by prolonged exposure to TCDD in vitro.     

Comparative studies on antitumor activity of Klebsiella O3 lipopolysaccharide and its polysaccharide fraction in mice

The antitumor activity of the polysaccharide fraction (OPS) obtained by the acid hydrolysis of Klebsiella O3 lipopolysaccharide (KO3 LPS) isolated from the culture supernatant of the decapsulated mutant strain LEN-1 (03: K1-) against both allogeneic tumor and syngeneic tumor systems in mice was compared with that of KO3 LPS. OPS prolonged the life span of MM2-bearing C3H/He mice by intraperitoneal (i.p.) pre- and post-treatment at the doses of 100 and 1000 mg/kg. However, large amounts of OPS were needed to show the antitumor activity as compared with KO3 LPS. OPS showed no growth inhibitory activity against Meth-A sarcoma in BALB/c mice by i.p., intravenous (i.v.) or intratumoral (i.t.) administration. When 1000 mg/kg of OPS was i.p. administered once a day for 10 days, OPS significantly inhibited the tumor growth of Sarcoma-180 solid type tumor. On the other hand, KO3 LPS significantly suppressed the growth of Meth-A tumor by i.t. administration at the doses of 0.3 and 1.0 mg/kg and showed complete regression in 8 and 9 out of 10 mice, respectively. In MM2 tumor, KO3 LPS also showed complete regression in all mice post-treated by i.p. administration at the dose of 1.0 mg/kg. These results suggest that OPS has antitumor activity on the tumors used in this study, but the activity was less than that of KO3 LPS.     

The effect of PARS inhibition on ileal histopathology, apoptosis and lipid peroxidation in LPS-induced obstructive jaundice.

In our experimental study, we investigated the protective effect of 3-aminobenzamide (3-AB), the poly (ADP-ribose) synthetase (PARS inhibitor), on the ileal histopathology and the apoptosis in lipopolysaccharide (LPS)-induced inflammation in rats with obstructive jaundice (OJ). We randomized 40 rats into five groups. Group 1: sham group; Group 2: OJ group; Group 3: OJ+LPS; Group 4: OJ+3-AB+LPS; Group 5: OJ+LPS+3-AB. At the fifth day; the rats were jaundiced. In Group 3; 10 mg kg(-1) LPS was injected intraperitoneally at the fifth day and then after 6h the rats were sacrificed. In Group 4; 10 mg kg(-1) 3-AB was administrated intraperitoneally at the fifth day and repeated daily for 3 days and at the eighth day, 10 mg kg(-1) LPS was injected intraperitoneally. In Group 5, 10 mg kg(-1) LPS was injected intraperitoneally at the fifth day and after 6h 10 mg kg(-1) 3-AB was administrated intraperitoneally and repeated daily for 3 days. At the eighth day, rats were sacrificed. Blood samples were taken for detection of serum MDA levels. Ileum samples were taken after relaparotomy for histopathological examination to evaluate the endotoxin-related intestinal injury and Caspase-3 apoptosis and for detection of tissue MDA and ATPase activities. There was marked destruction of villous and crypt epithelial cells and extensive apoptosis in Groups 3 and 5 in histopathological examination. In Group 4, the scores of intestinal mucosal damage and apoptotic cells were reduced significantly (P<0.05). On the other hand, the scores of intestinal mucosal damage and apoptotic cells were not improved in Group 5. After the administration of 3-AB (Group 4), serum and ileal MDA levels decreased, ileal ATPase increased as compared to Groups 1 and 2. Our study showed that 3-AB prevented the mucosal damage and apoptotic loss of intestinal epithelial cells significantly if it was administrated before LPS. However, 3-AB failed to prevent the mucosal damage and apoptotic loss of intestinal epithelial cells significantly if there was established endotoxemia in OJ.