Vitamin A deficiency and the induction of cutaneous toxicity in murine skin by TCDD

The mechanisms involved in the induction of toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototype for a group of toxic polyhalogenated aromatic hydrocarbons, are largely unknown. To test the hypothesis that TCDD-induced toxicity involves the reduction of vitamin A levels, we investigated the role of vitamin A deficiency in modulating the cutaneous response of congenic haired (+/+) and hairless (hr/hr) mice to TCDD. Hairless mice are recognized as sensitive models for expression of TCDD-induced cutaneous toxicity. Haired mice normally do not develop a cutaneous response to TCDD. Mice raised on a vitamin A-deficient diet, and age- and sex-matched controls raised on standard chow, were treated topically with TCDD and their cutaneous responses monitored histologically. Body weights and thymus gland weights were monitored as additional parameters of toxicity. Liver and skin vitamin A levels were determined by HPLC. Vitamin A depletion by itself had no effect on the normal cutaneous histology of the haired phenotype, nor were any changes in cutaneous morphology attributable to TCDD toxicity observed in haired, TCDD-treated animals even when they were severely vitamin A depleted. On the other hand, in hairless mice, vitamin A deficiency caused a distinct increase in keratinization of dermal epithelial cysts, and an increase in the sensitivity of these cysts to TCDD-induced hyperkeratinization. TCDD-induced body weight loss and atrophy of the thymus gland were not affected by the vitamin A status of either the haired or hairless animals. Analysis of vitamin A levels in skin and liver, following topical treatment of mice with TCDD, indicated that TCDD exposure did not affect cutaneous levels, but did significantly lower liver levels of vitamin A. These experiments suggest that although systemic vitamin A deficiency may potentiate the expression of TCDD-induced toxicity in skin of hairless mice, expression of TCDD-induced toxicity probably involves more complex mechanisms than a reduction in vitamin A levels.     

Increased epidermal transglutaminase activity following 2,3,7,8-tetrachlorodibenzo-p-dioxin: In vivo and in vitro studies with mouse skin

In previous studies it has been shown that topical treatment of hairless mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) induces hyperproliferation and hyperkeratinization in the epidermis of hairless mice. The present investigation demonstrated that such TCDD-induced morphological changes in skin in vivo are accompanied by increased levels in activity of epidermal transglutaminase (ETG), the enzyme associated with terminal epidermal differentiation. Exposure of mouse epidermal cells in tissue culture to 10^?9m TCDD also resulted in a significant increase in ETG activity, despite the fact that morphologically these cultures (grown at 0.07 mm ionic calcium concentrations) exhibited no signs of terminal differentiation. Thus one mechanism of action of TCDD in inducing cutaneous changes appears to relate to the stimulation of increased ETG levels.     

Response of murine epidermal keratinocyte cultures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): comparison of haired and hairless genotypes

Based on the observation that congenic haired and hairless mice differ in the hyperkeratinizing/hyperproliferative epidermal response following topical exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo, it has been proposed that this response in mice segregates with the Ah locus (which controls for the Ah receptors) and the hr locus (for hairlessness) (Knutson and Poland, 1982). In the present studies this hypothesis was tested by comparing the response of epidermal keratinocytes derived from genotypically segregated newborn haired and hairless HRS/J mice to TCDD exposure in in vitro cultures. the parameters monitored were stimulation of cell proliferation (as measured by protein content and rate of tritiated thymidine incorporation in cultures), epidermal transglutaminase activity, cornified cell envelope formation, and keratin staining with Rhodanile blue. Results suggested that the sensitivity of HRS/J haired and hairless mouse epidermal keratinocyte cultures to TCDD exposure in vitro was very similar. In both cell cultures all parameters monitored were stimulated by TCDD exposure in a similar dose-dependent manner. This suggests that physiologic factors beyond the epidermal cells may be involved in expression of the different responses seen in the skin of mice to TCDD in vivo.     

Pleiotropic effect of the gene hairless on hepatotoxicity of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin in mice

Treatment of mice of the A2G-hr/+ congenic line with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in the development of hepatic porphyria over a period of 4 weeks. Female mice responded to a lesser extent than did males. The degree of porphyria in haired heterozygotes (hr/+) was less than in the corresponding hairless homozygotes (hr/hr) and the haired mice had lower resting metabolic rates than hairless mice. Adaptation of mice of either genotype to a 32–33 °C environment resulted in a decrease in resting metabolic rate and a reduction in hepatic porphyrin levels. Histologically-demonstrated necrotic changes in livers were accompanied by increased activity of alanine aminotransferase and sorbitol dehydrogenase in the plasma; however, there was no clear temporal trend in plasma enzyme levels. Elevated environmental temperature reduced the plasma alanine aminotransferase activity. The study provided evidence for a pleiotropic effect of variation at the hr locus being expressed in TCDD hepatotoxicity. Suggestions for mechanisms whereby the effect can be mediated through alterations in resting metabolic rate are made.     

Inhibition of n-hexadecane-induced epidermal hyperplasia due to systemically administered ciclosporin

Epidermal hyperplasia was induced in hairless mice (hr/hr) by topical n-hexadecane treatment of tail and back skin. Following this skin irritation, a granular layer developed in interfollicular regions of the tail epidermis. An increase of ornithine decarboxylase activity, of thymidine triphosphate incorporation into DNA and of amino acid incorporation into protein was found. Shown histologically and by measurement of the called biochemical parameters, ciclosporin (cyclosporin A, CAS 59865-13-3; pretreatment with 30 mg/kg b.w. per day subcutaneously for 7 days) inhibited the development of epidermal hyperplasia in back and tail epidermis.     

Effects of src-deficiency on the expression of in vivo toxicity of TCDD in a strain of c-src knockout mice procured through six generations of backcrossings to C57BL/6 mice

The effect of TCDD was studied in c-src-deficient C57BL6-src(tm1sor) (N6 src -/- and -/+) mice, and their wild-type littermate mice (N6 src +/+). The former was created from the original strain of B6, 129-src(tm1sor) mice through six generations of backcrossings with C57BL6 mice. The results of a high dose TCDD toxicity tests in male mice indicated that N6 src-/+ mice were significantly less responsive to the toxic action of TCDD (115 microg/kg single i.p. injection) than N6 src+/+ mice in terms of reduced % body weight gain, the increase in the liver to body weight ratio, and the decrease in the adipose tissue to liver weight ratio and in the weight of pancreas. To understand the cause for these differential effects of TCDD we studied TCDD-induced changes in several biochemical parameters at day 10 and found that most drastically affected ones were glycogen depletion and phosphoenolpyruvate carboxykinase (PEPCK) downregulation. In addition, the degree of triglyceride accumulation in liver was less pronounced in N6-/+ than in N6+/+ mice. These findings suggest that the absence of c-src expression indeed affects the development of selected, TCDD-induced toxic endpoints that are related to wasting syndrome.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) enhances terminal differentiation of cultured human epidermal cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and isosteric halogenated analogs produce a spectrum of pathologic changes in the epidermis of humans. In this study, the actions of TCDD on cultured human epidermal cells were characterized to determine whether these cells are an appropriate in vitro model to examine the mechanisms of TCDD toxicity to human skin. The differential staining properties of TCDD-treated cultures indicated that TCDD decreased basal cell numbers and increased the degree of keratinization. Histologic examination of cross-sections of the cultures confirmed a loss of small nucleated cells and increased cell layering in response to TCDD. TCDD produced no change in total cell number or cell protein, but decreased the number of small (basal) cells and DNA synthesis. TCDD increased the number of cells containing spontaneous envelopes, as well as the number of envelope-competent cells. The quantitative changes observed in these parameters were consistent with a TCDD-induced commitment of proliferating cells to terminal differentiation. TCDD also decreased epidermal growth factor (EGF) specific binding. Maximal changes in EGF binding occurred after 4 days, and in small cell number after 5 days. The decreases in EGF binding and small cell number were stereospecific and concentration dependent (EC50, 1 to 2 nM), implicating the human Ah receptor in mediating these responses to TCDD. These data indicate that TCDD treatment produces hyperkeratinization in cultured human epidermal cells. It is proposed that TCDD acts on epidermal basal cells to enhance terminal differentiation through mechanisms regulated at least in part by the Ah receptor.     

Characteristics of 2,3,7,8-tetrachlorodibenzo-p-dioxin induced endotoxin hypersensitivity: association with hepatotoxicity

Hepatic clearance is a major route of endotoxin detoxification. In the present study, the potential relationship between TCDD-induced endotoxin hypersensitivity and hepatotoxicity was examined. Acute doses of 50, 100, or 200 micrograms TCDD/kg body weight induced an endotoxin hypersensitive state in B6C3F1 mice as demonstrated by increased mortality 24-48 h following i.v. injection of endotoxin. This hypersensitive state occurred when endotoxin was administered 7 days following TCDD exposure, but not 1 day post-TCDD exposure. TCDD did not affect endogenous serum endotoxin levels. However, clearance of injected endotoxin was significantly inhibited following exposure to TCDD. Six hours post endotoxin treatment serum triglycerides were significantly increased in TCDD/endotoxin-treated mice compared to either treatment alone. Methylprednisolone and uridine were both examined in this model due to their roles in inflammation and RNA synthesis, respectively. Both compounds significantly reversed the mortality associated with the combined exposure. [3H]Uridine incorporation into liver was decreased following TCDD treatment alone, further suggesting impaired RNA synthesis. Studies performed on congenic mice indicate that the observed effects segregate with the Ah locus. The ability of methylprednisolone and uridine to reverse the mortality associated with TCDD/endotoxin treatment is consistent with an inflammatory response and impaired hepatic detoxification mechanisms. Thus, changes in hepatic handling of endotoxin, caused by progressive TCDD-induced liver dysfunction, may be responsible for the endotoxin hypersensitivity.     

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on murine heart development: alteration in fetal and postnatal cardiac growth, and postnatal cardiac chronotropy.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related chemicals are potent cardiovascular teratogens in developing piscine and avian species. In the present study we investigated the effects of TCDD on murine cardiovascular development. Pregnant mice (C57Bl6N) were dosed with 1.5-24 microg TCDD/kg on gestation day (GD) 14.5. At GD 17.5, fetal mice exhibited a dose-related decrease in heart-to-body weight ratio that was significantly reduced at a maternal dose as low as 3.0 microg TCDD/kg. In addition, cardiocyte proliferation was reduced in GD 17.5 fetal hearts at the 6.0-microg TCDD/kg maternal dose. To determine if this reduction in cardiac weight was transient, or if it continued after birth, dams treated with control or 6.0 microg TCDD/kg were allowed to deliver, and heart weight of offspring was determined on postnatal days (P) 7 and 21. While no difference was seen on P 7, on P 21 pups from TCDD-treated litters showed an increase in heart-to-body weight ratio and in expression of the cardiac hypertrophy marker atrial natriuretic factor. Additionally, electrocardiograms of P 21 offspring showed that the combination of in utero and lactational TCDD exposure reduced postnatal heart rate but did not alter cardiac responsiveness to isoproterenol stimulation of heart rate. These results demonstrate that the fetal murine heart is a sensitive target of TCDD-induced teratogenicity, resembling many of TCDD-induced effects observed in fish and avian embryos, including reduced cardiocyte proliferation and altered fetal heart size. Furthermore, the combination of in utero and lactational TCDD exposure can induce cardiac hypertrophy and bradycardia postnatally, which could increase the risk of cardiovascular disease development.     

TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin.     

Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR)

The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (− 105/+ 1 base pair). Fgf21-null mice administered 200 μg/kg of TCDD died within 20 days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver.     

Relative toxicity and tumor-promoting ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PCDF), and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) in hairless mice

2,3,7,8-Tetrachlorodibenzo-p-dixoin 2,3,4,7,8-pentachlorodibenzofuran (PCDF), and 1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) are highly toxic members of a class of environmental contaminants, the polychlorinated aromatic hydrocarbons (PCAH), which exhibit a similar and highly characteristic spectrum of toxic effects. For purposes of risk assessment, it is important to be able to make accurate estimates of the relative potency of these and related compounds. Previous investigations have indicated that, in acute exposure or in vitro studies, PCDF is approximately 0.1 times as toxic and HCDF is approximately 0.01 times as toxic as TCDD. In this study, we compared the relative toxicity and tumor-promoting abilities of TCDD, PCDF, and HCDF in hairless mouse skin. Female hairless mice (HRS/J hr/hr) were treated dermally with the initiator MNNG, then dosed twice weekly for 20 weeks with acetone, TCDD (2.5-10 ng/mouse/dose), PCDF (25-100 ng/mouse/dose), or HCDF (250-1000 ng/mouse/dose) as promoter. TCDD, PCDF, and HCDF were all potent promoters for the induction of squamous cell papillomas. There was, however, no difference in the incidence or multiplicity of papilloma formation between groups. The same doses of the three PCAH, in the absence of initiator, induced no skin papillomas. TCDD produced a significant increase in liver:body weight ratio (p less than 0.001) at all doses and a decrease in thymus:body weight ratio at a dose of 10 ng (p less than 0.001). Mice treated with PCDF and HCDF had marked thymic and splenic involution, liver hypertrophy, mucous cell hyperplasia in the fundic portion of the glandular stomach, and loss of body weight. PCDF and HCDF produced a greater incidence and severity of dermatotoxic effects than TCDD. Based on data for dermal toxicity and changes in body weight and organ weights, PCDF is estimated to be 0.2 to 0.4 times, and HCDF 0.08 to 0.16 times, as toxic as TCDD following repeated dermal exposure. Therefore, toxic equivalence factors generated using data from acute and/or in vitro studies may underestimate the risk from repeated low-dose exposures to these compounds.     

Oral and dermal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cutaneous papillomas and squamous cell carcinomas in female hemizygous Tg.AC transgenic mice.

Tg.AC mice develop epidermal papillomas in response to treatment with dermally applied nongenotoxic and complete carcinogens. The persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a multi-site rodent carcinogen and tumor promoter that induces the formation of papillomas in Tg.AC mice. To examine the dose-response relationship and compare dermal and oral routes of exposure for TCDD-induced skin papillomas, female Tg.AC mice were exposed dermally to average daily doses of 0, 2.1, 7.3, 15, 33, 52, 71, 152, and 326 ng TCDD/kg/day or 0, 75, 321, and 893 ng TCDD/kg body weight by gavage for 26 weeks. The incidence of cutaneous papillomas was increased in a dose-dependent manner, and tumors developed earlier with higher exposure to TCDD regardless of route of administration. Increased incidences of cutaneous squamous cell carcinomas were observed in mice exposed to dermal (> or =52 ng/kg) and oral (893 ng/kg) TCDD. Higher gavage doses than dermal exposure doses were required to induce papillomas and squamous cell carcinomas. Despite a linear correlation between administered dose and terminal skin concentrations, the incidence of tumor formation was lower in the gavage study than in the dermal study with respect to mean terminal skin TCDD concentrations. These studies demonstrate that, although Tg.AC mice are less responsive to TCDD by gavage than by dermal exposure, the induction of skin neoplasms is a response to systemic exposure and not solely a local response at the site of dermal application. Differences in response between the routes of exposure may reflect pharmacokinetic differences in the delivery of TCDD to the skin over the duration of the study.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced porphyria in genetically inbred mice: partial antagonism and mechanistic studies

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (233 nmol/kg) causes a significant increase of hepatic uroporphyrin, heptacarboxyporphyrin, and total porphyrins in female C57BL/6 mice, ovariectomized C57BL/6 mice, male C57BL/10 mice, and male C57BL/6 mice 3 weeks after treatment. In contrast, 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) was inactive at a dose of 750 mumol/kg. Cotreatment of the mice with TCDD (233 mol/kg) plus MCDF (750 mumol/kg) resulted in partial antagonism of TCDD-induced hepatic porphyrin accumulation only in the female mice. Parallel studies in female C57BL/6 mice showed that the TCDD-induced porphyria was accompanied by the induction of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) activities and the depression of uroporphyrinogen decarboxylase (UROD). MCDF (750 mumol/kg) did not significantly affect these enzymes. In the cotreatment studies (MCDF plus TCDD), MCDF partially antagonized TCDD-induced hepatic porphyrin accumulation but did not affect the levels of hepatic AHH, EROD, or UROD. These results indicate that other factors, in addition to the induction of cytochrome P450-dependent monooxygenases and depressed UROD activity, are important in TCDD-induced porphyria in C57BL/6 female mice.     

The hr locus and the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in newborn mice

 In mice, the recessive mutation hairless (hr) controls the cutaneous response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but its influence on TCDD’s systemic toxicity is unclear. To clarify this, we compared the effects of lactational TCDD exposure on standardized litters of newborn HRS/J mice homozygous for either hr or + that were fostered by haired dams exposed to 0, 6, 8 or 12 μg TCDD/kg body weight on postnatal day 0. At 12 μg/kg, TCDD was lethal to both haired and hairless pups. At the lower doses (6 and 8 μg/kg) the survival of hr/hr pups was significantly lower than +/+ pups. Affected pups succumbed following a 1 to 2-day period of cachexia and wasting. As has been reported for other mouse strains, TCDD exposure impacted on their neonatal development and lessened the time to eye opening for both haired and hairless pups. However, the hairless animals were affected at lower doses than were the haired. The results of this study document that the hr/hr genotype does influence the systemic toxicity of TCDD in mice. Received: 26 April 1994 / Accepted: 23 June 1994     

Tumor necrosis factor involvement in 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated endotoxin hypersensitivity in C57BL/6J mice congenic at the Ah locus.

An experimental model of endotoxin-induced release of tumor necrosis factor-alpha (TNF) into the serum of C57BL/6J mice congenic at the Ah locus was used to investigate the effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on TNF production. TCDD exposure of Ah-responsive mice (Ahbb) resulted in a dose-dependent increase in the concentration of TNF in the serum of endotoxin-exposed mice, with a significant increase observed at a dose of 10 micrograms/kg TCDD. At a dose of 500 micrograms/kg TCDD, Ahbb mice demonstrated a 46-fold increase in serum TNF levels compared to control. In contrast, congenic Ah-receptor deficient mice (Ahdd) did not show a significant increase in serum TNF levels until exposed to 150 micrograms/kg TCDD, and the maximum response was an 8-fold increase over control. These data suggest that increased TNF production may be responsible for endotoxin hypersensitivity in TCDD-treated mice and that the Ah locus mediates this response.     

Hairless mice as models for chloracne: a study of cutaneous changes induced by topical application of established chloracnegens

Two types of hairless mice, one from the inbred strains, $\text{HRS}\text{J}$, and the other from the outbred stock, Skh:HR-1, were used to study the feasibility of inducing experimental chloracne with various well-established chloracnegens. Animals were treated topically for periods ranging from 2 to 10 weeks, and the cutaneous changes were monitored. Most chloracnegens induced a distinct pattern of cutaneous pathology, but this pattern did not include hyperkeratinization of the sebaceous follicles, which is considered the pathognomonic lesion in human chloracne. Rather, the changes induced by chloracnegens such as Halowax 1014, N-wax-34, Phenclor 54, and 2,3,4,8-tetrachlorodibenzo-p-dioxin were: epidermal hyperkeratosis and hyperplasia, loss of sebaceous glands, keratinization of intradermal pilar cysts, and, often, diffuse lymphohistiocytic infiltration of the dermis. Only one chloracnegen, 3,4,3′,4′-tetrachlorobiphenyl, induced a true chloracne-like syndrome, and only in one of the strains of mice (Skh:HR-1). This syndrome involved massive hyperkeratosis of the sebaceous follicles and hyperkeratinization of intradermal pilar cysts. In many instances, these cysts spontaneously ruptured into the dermis and induced the development of pimple-like foci of inflammatory cell accumulation in the skin. All animals developing these cutaneous changes also underwent a generalized metabolic disturbance resulting in gross weight gain, primarily due to large intraabdominal fat deposits.     

CYP1A2 is not required for 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced immunosuppression

One of the most sensitive and reproducible immunotoxic endpoints of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure is suppression of the antibody response to sheep red blood cells (SRBCs) in mice. Immunosuppression occurs in concert with hepatomegaly and associated induction of several hepatic cytochrome P450 enzymes, including CYP1A2 which is responsible for the hepatic sequestration of TCDD. In this study, TCDD-induced immunosuppression was evaluated in C57BL/6N CYP1A2 (+/+) wild-type and compared with that of age-matched CYP1A2 (-/-) knockout and CYP1A2 (+/-) heterozygous female mice. Groups of mice were given a single gavage dose of 0, 0.03, 0.1, 0.3, 1.0, 3.0 or 10.0microg TCDD/kg, followed 7 days later by immunization with SRBCs. Serum was obtained 5 days after immunization and body, spleen, thymus and liver weights were measured. sheep red blood cell (SRBC) antibody titers were determined by an enzyme-linked immunosorbent assay (ELISA). Anti-SRBC titers were suppressed at 1.0, 1.0 and 0.3microg TCDD/kg for CYP1A2 (+/+), CYP1A2 (+/-), and CYP1A2 (-/-) mice, respectively, which indicated a three-fold increase in TCDD-induced immunosuppression for the CYP1A2 (-/-) mice. This increase in TCDD-induced immunosuppression may be due to the inability of CYP1A2 (-/-) mice to sequester TCDD in the liver leading to a higher dose to the immune system. In CYP1A2 (+/+) mice, a dose of 3.0microg TCDD/kg was sufficient to increase the liver weight, while in CYP1A2 (-/-) mice no increase in liver weight was observed. Application of analysis of variance and dose-response modeling approaches indicate that there is little evidence that the immunosuppression dose-response curves, for the three strains, differ in the lower part of the dose-response range. Thus, CYP1A2 is not required for TCDD-induced immunosuppression in the mouse.     

Mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced decrease in anti-CD3-activated CD4(+) T cells: the roles of apoptosis, Fas, and TNF.

The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), suppresses T cell functions and reduces T cell numbers in multiple models of immune stimulation. However, the underlying mechanism(s) by which TCDD induces these changes has yet to be elucidated. We hypothesized that TCDD affects T cells through the induction or augmentation of apoptosis. In these studies, we used antibody to CD4, annexin V, and 7-AAD in three-color flow cytometric analyses to examine the relationship between the decrease in CD4(+) T cells and cell death in mice treated with anti-CD3 and TCDD. In addition, we examined two signaling pathways, Fas and TNF, in order to elucidate a potential mechanism by which TCDD increases cell death. Our results show that the TCDD-induced decrease in CD4(+) T cell number correlated with an increase in the percentage of dead cells, but not with cells expressing an early apoptotic phenotype. The TCDD-induced decrease in CD4(+) T cells was attenuated in Fas- and FasL-deficient mice (lpr and gld, respectively), but not by treatment with a neutralizing antibody to TNF. While these results suggest that the Fas pathway may be important in TCDD-induced T cell death, however, the effect of TCDD on the Fas pathway remains unclear. Taken together, our data suggest that TCDD-induced suppression of CD4(+) T cells involves, in part, increased cell death that may be mediated by Fas/FasL interaction.