Effect of subchronic 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on immune system and target gene responses in mice: calculation of benchmark doses for CYP1A1 and CYP1A2 related enzyme activities

The dose-effect relationships were analysed for several noncarcinogenic endpoints, such as immunological and biochemical responses at subchronic, low dose exposure of female C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The animals were treated i.p. with TCDD according to the initial- and maintenance-dose principle for a period of 135 days. The initial doses were 1, 10 and 100 ng TCDD/kg, the weekly maintenance doses were 0.2, 2 and 20 ng TCDD/kg, respectively. At days 23, 79 and 135 of TCDD treatment 10 animals of each dose group were killed. As immunological parameters the number of thymocytes and the pattern of thymocyte subpopulations were determined. In liver, lung and thymus, mRNA expression of TGF-α, TGF-β₁, TGF-β₂, TGF-β₃, TNF-α, IL-1β and different CYP1 isoforms (CYP1A1, CYP1A2, CYP1B1) was analysed. In the livers, activities of 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) were measured. TCDD content in the liver was determined. The main results are summarized as follows: (1) The TCDD doses were not sufficient to elicit dose-dependent changes of pattern of thymocyte subpopulation. (2) TCDD failed to change the mRNA expression of TGF-α, TGF-β and TNF-α, but led to an increase of IL-1β mRNA expression in liver, lung and thymus. The results show that the TCDD induced IL-1β mRNA increase is at least as sensitive a marker as the induction of CYP1A isoforms. (3) The expression of CYP1B1 mRNA remained unchanged at the doses tested, while CYP1A1 and CYP1A2 mRNA expression was dose-dependently enhanced. EROD and MROD activities in the liver paralleled the increases of CYP1A1 and CYP1A2 mRNA expression. (4) Regression analysis of the data showed that most of the parameters tested fit a linear model. (5) From the data, a benchmark dose for EROD/MROD activities in the livers of female C57BL/6 mice of about 0.03 ng TCDD/kg per day was calculated. Received: 7 October 1996 / Accepted: 20 November 1996     

Use of Model-Based Compartmental Analysis to Study Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Vitamin A Kinetics in Rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic,widespread environmental contaminant that has dramatic adverseeffects on the metabolism of vitamin A. We used model-basedcompartmental analysis to investigate sites and quantitativeimpacts of TCDD on vitamin A kinetics in rats given an oralloading dose of TCDD in oil (3.5 µg/kg) followed by weeklymaintenance doses (0.7 µg/kg) or oil only. [³H]retinylin its plasma transport complex (experiment 1) or lymph containingchylomicrons labeled mainly with [³H]retinyl esters (experiment2) were administered iv, and tracer kinetics in plasma, liver,carcass, urine, and feces were measured for up to 42 days. TCDDtreatment caused significant reductions in liver vitamin A levelsand significant changes in tracer kinetics and tracer excretion.A four-compartment model was used to fit tracer data for experiment1; for experiment 2, compartments were added to describe themetabolism of newly absorbed vitamin A. The compartmental modelspredict that TCDD caused a slight delay in plasma clearance(via an increased recycling to plasma), and in liver processing,of chylomicronderived vitamin A. Models for both experimentspredict that TCDD exposure did not affect the fractional uptakeof plasma retinol from the rapidly turning-over extravascularpool, but it doubled the fractional transfer of recycled retholfrom slowly turning-over pools of vitamin A to plasma The residencetime for vitamin A was reduced by 70% in TCDD-treated rats,transfer into urine and feces was tripled, and vitamin A utilizationrates were significantly increased. Since our results do notindicate that retino1 esterification is inhibited, we hypothesizethat some of the significant effects of TCDD on vitamin A metabolismresult from increased catabolism and mobilization of vitaminA from slowly turning-over pools (especially the liver).     

Suppression of CYP1A1 expression by naringenin in murine Hepa-1c1c7 cells

Naringenin, dietary flavonoid, is antioxidant constituents of many citrus fruits. In the present study, we investigated the effect of naringenin on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible CYP1A1 gene expression in mouse hepatoma Hepa-1c1c7 cells. Naringenin alone did not affect CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity. In contrast, the TCDD-inducible EROD activities were markedly reduced upon concomitant treatment with TCDD and naringenin in a dose dependent manner. TCDD-induced CYP1A1 mRNA level was also markedly suppressed by naringenin. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and electrophoretic mobility shift assay revealed that naringenin reduced transformation of the aryl hydrocarbons receptor(AhR) to a form capable of specifically binding to the DRE sequence in the promoter of the CYP1A1 gene. These results suggest the down regulation of the CYP1A1 gene expression by either naringenin in Hepa-1c1c7 cells might be antagonism of the DRE binding potential of nuclear AhR.     

Low Inducibility of CYP1A Activity by Polychlorinated Biphenyls (PCBs) in Flounder (Platichthys flesus): Characterization of the Ah Receptor and the Role of CYP1A Inhibition

Several studies have reported a low inducibility of hepaticcytochrome P4501A (CYP1A) activity in European flounder (Platichthysflesus) following exposure to mixtures of polychlorinated biphenyls(PCBs). Here we report on mechanistic studies toward understandingthis low CYP1A inducibility of flounder, involving molecularcharacterization of the Ah receptor (AhR) pathway as well asinhibition of the CYP1A catalytic activity by PCB congeners.Hepatic cytosolk AhR levels in flounder were determined usinghydroxylapatite, protamine sulfate adsorption analysis, or velocitysedimentation on sucrose gradients. AhR levels in flounder ({smalltilde}2–7 fmol/mg protein) were much lower than observedgenerally in rodents ({small tilde}50–300 fmol/mg protein).Molecular characterization of the flounder AhR was providedby first-strand cDNA synthesis and amplification of flounderhepatic poly(A)⁺ RNA using RT-PCR. A 690-bp product was found,similar in size to a Fundulus AhR cDNA. The specificity of the690-bp band was established by Southern blotting and hybridizationwith a degenerate AhR oligonucleotide. The deduced amino acidsequence of the flounder AhR fragment was 59–60% identicalto mammalian AhR sequences. Although the AhR is present in floundercytosol, we were unable to demonstrate detectable amounts ofinducibk TCDD-AhR-DRE complex in gel-retardation assays. Highinduction levels of CYP1A protein and associated EROD activityhave been previously found in flounder following exposure to2,3,7,8-tetrachlorod-ibenzo-p-dioxin (TCDD). In contrast, theinduction of CYP1A catalytic activity by PCB mixtures remainsunexpectedly low. Therefore, we further characterized the inhibitorypotential of PCB congeners on CYP1A activity in flounder andcompared this with inhibitory effects of PCB congeners on ratCYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB,3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB,and the commercial PCB mixture Clophen A50 are potent competitiveinhibitors of hepatic microsomal CYP1A catalytic activity inflounder and rat The K_m for ethoxyresonifin (0.095 µM)in flounder is strikingly close to K_i's found for the testedPCBs. This emphasizes the possible involvement of PCB congenersin inhibition of EROD activity in PHAH exposed fish. Finally,our data indicate that flounder CYP1A is more efficient in metabolizingethoxyresonifin than that of rat CYP1A.     

Altered thyroxin and retinoid metabolic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin in aryl hydrocarbon receptor-null mice

To determine whether the disruption of thyroid hormone and retinoid homeostasis that occurs after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated by the arylhydrocarbon receptor (AhR), pregnant AhR-heterozygous (AhR+/–) mice were administered a single oral dose of 10 g kg^–1 TCDD at gestation day 12.5. Serum and liver were collected on postnatal day 21 from vehicle-treated control or TCDD-treated AhR+/– and AhR-null (AhR–/–) mouse pups. Whereas TCDD exposure resulted in a marked reduction of total thyroxin (TT4) and free T4 (FT4) levels in the serum of AhR+/– mice, TCDD had no effects on AhR–/– mice. Gene expression of UDP-glucuronosyltransferase (UGT)1A6, cytochrome P450 (CYP)1A1, and CYP1A2 in the liver was induced markedly by TCDD in AhR+/– but not AhR–/– mice. Induction of CYP1A1 in response to TCDD was confirmed by immunohistochemical evidence in that CYP1A1 protein was conspicuously localized in the cytoplasm of hepatocytes in the centrilobular region. Levels of retinyl palmitate were greatly reduced in the liver of TCDD-exposed AhR+/– mice, but not in vehicle-treated AhR+/– mice. No effects of TCDD on retinoid levels in the liver were found in AhR–/– mice. We conclude that disruption of thyroid hormone and retinoid homeostasis is mediated entirely via AhR. Induction of UGT1A6 is thought to be responsible at least partly for reduced serum thyroid hormone levels in TCDD-exposed mice.     

Superinduction of CYP1A1 in MCF10A cultures by cycloheximide, anisomycin, and puromycin: a process independent of effects on protein translation and unrelated to suppression of aryl hydrocarbon receptor proteolysis by the proteasome

Exposure of the human breast epithelial cell line MCF10A to > or = 1 microg/ml cycloheximide (CHX)-induced accumulations of CYP1A1 mRNA 6-fold greater than that achieved with only 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cotreatment with CHX and TCDD caused superinduction of CYP1A1 with accumulations of CYP1A1 mRNA 30-fold greater than that achieved with only TCDD. Similar results were obtained with the protein translation inhibitors anisomycin (ANS) and puromycin (PUR). Intra- and interinhibitor comparisons of dose/concentration response curves demonstrated the absence of a quantitative relationship between [3H]leucine incorporation and CYP1A1 induction/superinduction. The inducing/superinducing activities of CHX were suppressed by coincubation with the aryl hydrocarbon receptor (AhR) antagonists alpha-naphthoflavone and 3'-methoxy-4'-nitroflavone (PD168641). Electrophoretic mobility shift assays demonstrated that nuclear extracts from CHX-treated and CHX + TCDD cotreated cultures formed approximately 58 and approximately 340% of the AhR/DNA complexes obtained with TCDD-treated cultures, respectively. In contrast, rat liver extracts did not form AhR/DNA complexes after in vitro transformation with CHX. AhR turnover in TCDD-treated hepatoma 1c1c7 cultures was suppressed by cotreatment with CHX. In contrast, CHX or ANS treatment of MCF10A cultures induced AhR loss and enhanced AhR loss in cultures cotreated with TCDD. Cotreatment with N-benzoyloxycarbonyl-(Z)-Leu-Leu-leucinal (MG132) but not leptomycin B suppressed AhR loss. Hence, in MCF10A cells, CHX is not an AhR agonist but can superinduce CYP1A1 via an AhR-dependent mechanism; CYP1A1 superinduction by translation inhibitors is neither quantitatively related to effects on protein synthesis nor due to a generalized prevention of AhR proteolysis, and proteasome-mediated degradation of the activated AhR can occur in the nucleus.     

Nrf2 protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced oxidative injury and steatohepatitis

Previous studies demonstrate that Nrf2, a master regulator of antioxidative responses, is essential in mediating induction of many antioxidative enzymes by acute activation of the AhR. However, the role of Nrf2 in protecting against oxidative stress and DNA damage induced by sustained activation of the AhR remains unknown and was investigated herein. Tissue and blood samples were collected from wild-type (WT) and Nrf2-null mice 21 days after administration of a low-toxic dose (10 μg/kg ip) of TCDD. Only Nrf2-null mice lost body weight after TCDD treatment; however, blood levels of ALT were not markedly changed in either genotype, indicating a lack of extensive necrosis. Compared to livers of TCDD-treated WT mice, livers of TCDD-treated Nrf2-null mice had: 1) degenerated hepatocytes, lobular inflammation, marked fat accumulation, and higher mRNA expression of inflammatory and fibrotic genes; 2) depletion of glutathione, elevation in lipid peroxidation and marker of DNA damage; 3) attenuated induction of phase-II enzymes Nqo1, Gsta1/2, and Ugt2b35 mRNAs, but higher induction of cytoprotective Ho-1, Prdx1, Trxr1, Gclc, and Epxh1 mRNAs; 4) higher mRNA expression of Fgf21 and triglyceride-synthesis genes, but down-regulation of bile-acid-synthesis genes and cholesterol-efflux transporters; and 5) trend of induction/activation of c-jun and NF-kB. Additionally, TCDD-treated Nrf2-null mice had impaired adipogenesis in white adipose tissue. In conclusion, Nrf2 protects livers of mice against oxidative stress, DNA damage, and steatohepatitis induced by TCDD-mediated sustained activation of the AhR. The aggravated hepatosteatosis in TCDD-treated Nrf2-null mice is due to increased lipogenesis in liver and impaired lipogenesis in white adipose tissue.     

MicroRNA changes associated with atypical CYP1A1 inducer BMS-764459

The corticotrophin releasing factor (CRF) receptor I antagonist, BMS-764459 (evaluated as a potential treatment of affective disorders), was orally dosed to female Sprague-Dawley rats once daily for 2 weeks (vehicle control or 175 mg/kg/day). To investigate the mechanism of BMS-764459-related liver weight increases, total liver RNA was isolated and evaluated for mRNA gene expression by microarray analysis (assessing the expression of approximately 24,000 genes) from snap-frozen tissue. Subsequently, mRNA and miRNA (microRNA) were also analyzed 5 years later from FFPE (Formalin Fixed Paraffin Embedded) samples via RT-PCR (about 800 miRNA evaluated). Genomic analyses showed that BMS-764459 induces AhR target genes with additional inductions of CYP2B, CYP3A, and Abcc3 consistent with the gene expression pattern of atypical CYP1A1 inducers. Analysis of miRNA expression identified a number of significantly affected miRNAs. To further evaluate their role in atypical CYP1A1 induction, an in silico evaluation of differentially expressed miRNA was performed and their putative mRNA 3′-UTR (untranslated region) binding sequences were evaluated. MiR-680 and miR-29a were identified as potential regulators and biomarkers of atypical CYP1A1 induction by regulating Abcc3, CYP3A and CYP2B as well as a number of AhR targeted genes.     

Effect of biochanin A on the aryl hydrocarbon receptor and cytochrome P450 1A1 in MCF-7 human breast carcinoma cells

Phytoestrogen biochanin A is an isoflavone derivative isolated from red clover Trifolium pratense with anticarcinogenic properties. This study examined the action of biochanin A with the carcinogen activation pathway that is mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 breast carcinoma cells. Treating the cells with biochanin A alone caused the accumulation of CYP1A1 mRNA and an increase in CYP1A1-specific 7-ethoxyresorufin O-deethylase (EROD) activity in a dose dependent manner. A concomitant treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and biochanin A markedly reduced the DMBA-inducible EROD activity and CYP1A1 mRNA level. In addition, the biochanin A treatment alone activated the DNA-binding capacity of the AhR for the dioxin-response element (DRE) of CYP1A1, as measured by the electrophoretic-mobility shift assay (EMSA). EMSA revealed that biochanin A reduced the level of the DMBA-inducible AhR-DRE binding complex. Furthermore, biochanin A competed with the prototypical AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), for binding to the AhR in an isolated rat cytosol. The biochanin A competitively inhibited the metabolic activation of DMBA, as measured by the formation of the DMBA-DNA adducts. These results suggest that biochanin A may thus be a natural ligand to bind on AhR. Therefore, biochanin A may be due to act an antagonist/agonist of the AhR pathway.     

昆明种小鼠细胞色素氧化酶CYP1A基因表达的昼夜节律变化

目的探究昆明种小鼠细胞色素氧化酶1A1(CYP1A1)的昼夜节律、性别差异以及对肝毒物毒性变化的影响。方法昆明小鼠在环境控制的SPF饲养室内适应性饲养2周后,于06:00,10:00,14:00,18:00,22:00和次日02:00处死取肝,用逆转录PCR方法检测24h内核激素孤儿受体α(Rev-erbα),时钟基因(Per)1,Per2和CYP1A1,CYP1A2及其调控核受体基因AhR的表达。另取小鼠于6:00和18:00 ip给予对乙酰氨基酚500mg·kg-1,12h后检测血清中丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)活性。结果细胞色素氧化酶基因CYP1A1,CYP1A2及其调节基因AhR在18:00左右表达最高,且雌鼠的表达高于雄鼠,在6:00左右表达最低,表达峰谷差为4～7倍,其节律变化的差异与Rev-erbα,Per1,Per2的节律差异大致相符。与此节律相对应,对乙酰氨基酚引起的肝毒性在18:00给药高于6:00给药。结论昆明种小鼠细胞色素氧化酶CYP1A1基因表达存在昼夜节律及性别差异,该差异可影响肝毒物如对乙酰氨基酚的代谢和毒性。     

Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium.

Endometriosis is a debilitating disease estimated to affect 10% of reproductive-age women and characterized by the growth of endometrial tissue outside of the uterus. The present study characterizes a human endometrial explant culture model for studying the direct effects of TCDD exposure by assessing the expression of CYP1A1 and CYP1B1 mRNA (Northern blotting), protein (Western blotting), and activity (7-ethoxyresorufin-O-deethylase; EROD) in explants cultured with and without TCDD. Explants were obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. The explants were cultured with 10 nM estradiol (E(2)) or 1 nM E(2) plus 500 nM progesterone (P(4)) with or without TCDD (first 24 h). The expression of CYP1A1 and CYP1B1 mRNA was greatest with 10 nM TCDD and increased up to 72 h after initial exposure. EROD activity increased up to 120 h. Explants from a secretory phase biopsy became reorganized in culture and formed a new epithelial membrane, while maintaining basic endometrial morphology and viability for up to 120 h. At 24 h, TCDD significantly increased CYP1A1 and CYP1B1 mRNA, and at 72 h, TCDD significantly increased EROD activity and CYP1B1 protein compared to explants cultured without TCDD for similar times. CYP1B1 protein also exhibited substantial constitutive expression that was similar in uncultured biopsies, where CYP1B1 protein was immunolocalized in the cytoplasm of epithelial glands, with only occasional patches of protein in the surface epithelial membrane. In explants cultured with and without TCDD exposure, CYP1B1 protein was localized in the cytoplasm of the new surface epithelial membrane and glands closest to the surface. CYP1A1 protein was not detected in uncultured biopsies or explants. Both younger age (age 30 and under) and proliferative phase were associated with higher TCDD-induced EROD activity in specimens treated with E(2):P(4). No significant endometriosis-related differences were observed for any of the biomarkers, but the detection of disease-specific change was limited by small sample size and variability in tissue-cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.     

Hypophagia-Induced Weight Loss in Mice, Rats, and Guinea Pigs Treated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Hypophagia-lnduced Weight Loss in Mice, Rats, and Guinea PigsTreated with 2,3,7,8-Tetrachlorodibenzo-p-dioxin. KELLING, C.K, CHRISTIAN, B. J., INHORN, S. L., AND PETERSON, R. E. (1985).Fundam. Appl. Toxicol. 5, 700–712. C57BL/6 mice treatedwith 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 360 µ/kg)displayed a significant reduction in feed intake and body weightuntil just before death, when they developed ascites and subcutaneousedema. This caused body weight of the mice that died to suddenlyincrease during the terminal stage of toxicity. TCDD-treatedmice that survived did not develop ascites or edema, and maintaineda body weight that was slightly less than that of pair-fed mice.Cumulative lethality in TCDD-treated mice (69%) was greaterthan that of pair-fed controls (14%). In guinea pigs treatedwith TCDD (2 µ/kg) both the time course and magnitudeof hypophagja were closely associated with weight loss. Pair-fedguinea pigs did not lose quite as much weight as TCDD-treatedanimals because their total body water content was higher. Waterintake in pair-fed guinea pigs was greater than that of TCDD-treatedanimals. The time course and magnitude of lethality tended tobe similar in TCDD-treated guinea pigs (81%) and pair-fed controls(64%). In Fischer F-344 rats treated with TCDD (100 µg/kg)body weight loss was associated with a reduction in both feedand water intake. The time course and magnitude of weight lossin TCDD-treated and pair-fed rats was essentially identical.Lethality was higher in TCDD-treated rats (95%) than pair-fedcontrol animals (48%). Taken together, these findings suggestthat hypophagia is responsible for the loss of adipose and leantissue in mice, guinea pigs, and rats treated with a LD70–95dose of TCDD. Under these dosage conditions, weight loss contributesmore to the lethality of guinea pigs than to that of FischerF-344 rats or C57BL/6 mice     

Tissue-specific induction of cytochromes P450 1A1 and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in engineered C57BL/6J mice of arylhydrocarbon receptor gene

Tissue-specific induction of mRNA of cytochrome P450 (P450 or CYP) 1A1 and 1B1 by polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) was investigated in wild and arylhydrocarbon receptor (AhR)-deficient C57BL/6J mice. Ratios of mRNA expression of CYP1A1 or CYP1B1 over beta-actin were determined and used to compare levels of expression and induction of these P450s by PAHs and PCBs in various organs. CYP1A1 mRNA was detected in control mice at very low levels in liver, lung, heart, kidney, intestine, thymus, testis, uterus, ovary, and brain and was highly induced in these organs by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. In AhR(+/+) and AhR(-/-) mice, CYP1B1 mRNA was found to be constitutively expressed at significant levels in heart (the ratio of mRNAs of CYP1B1 to beta-actin was approximately 0.6), kidney ( approximately 0.8), intestine ( approximately 0.3), testis ( approximately 0.9), thymus ( approximately 0.4), uterus ( approximately 0.3), ovary ( approximately 1.4), and brain ( approximately 0.4), whereas it was low in liver and lung (the mRNA ratio to beta-actin was <0.2 in these cases). CYP1B1 in the latter two organs was highly induced by PAHs and 3,4,3',4'-tetrachlorobiphenyl in AhR(+/+) mice. The induction of CYP1B1 by PAHs and PCBs was more extensive in organs in which the constitutive expression of CYP1B1 was low. For example, CYP1B1 was induced 9-fold and 10-fold by benzo[a]pyrene and 3,4,3',4'-tetrachlorobiphenyl in livers of male and female mice, respectively, whereas in testis and ovary, the fold induction of CYP1B1 by two inducers was only 1.1 and 1.4, respectively. Liver microsomal xenobiotic oxidation activities were induced by these PAHs and PCBs in male and female AhR(+/+) mice. These results suggest that CYP1A1 and CYP1B1 are differentially regulated in their expression in extrahepatic organs of mice and could be induced by PAHs and PCBs with different extents of induction depending on the inducers used and the organs examined in AhR(+/+) mice. The findings of significant levels of constitutive expression of CYP1B1 in AhR(-/-) mice as well as AhR(+/+) mice in several organs including heart, kidney, thymus, testis, ovary, and brain in AhR(-/-) mice as well as AhR(+/+) mice are of importance in understanding the basis of toxicity and carcinogenesis by chemicals that are metabolized by CYP1B1.