Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish

The mechanism of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is thought to result from changes in gene expression via the aryl hydrocarbon receptor (AHR). The induction of cytochrome P450 1A (CYP1A) in various organs is a cardinal effect of TCDD. However, whether CYP1A is involved in endpoints of TCDD toxicity is controversial. We investigated the role of CYP1A in TCDD-induced developmental toxicities using gene knock-down with morpholino antisense oligos. Exposure of zebrafish embryos to TCDD, at concentrations eliciting the hallmark endpoints of developmental toxicity, induced CYP1A in the heart and vascular endothelium throughout the body. This induction by TCDD was markedly inhibited by morpholinos to zebrafish arylhydrocarbon receptor 2 (zfAHR2-MO) and to zebrafish CYP1A (zfCYP1A-MO). The zfAHR2-MO but not the zfCYP1A-MO inhibited zfCYP1A mRNA expression, indicating the specificities of these morpholinos. Injection of either zfAHR2-MO or zfCYP1A-MO blocked the representative signs of TCDD developmental toxicity in zebrafish, pericardial edema and trunk circulation failure. The morpholinos appeared do not affect normal development in TCDD-untreated embryos. These results suggest a mediatory role of zfCYP1A induction through zfAHR2 activation in causing circulation failure by TCDD in zebrafish. This is the first molecular evidence demonstrating an essential requirement for CYP1A induction in TCDD-evoked developmental toxicities in any vertebrate species.     

Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain

The aryl hydrocarbon receptor repressor (AHRR) is a negative regulator of AH receptor (AHR), which mediates most of the toxic and biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has been shown to be the major reason for the exceptionally wide (ca. 1000-fold) sensitivity difference in acute toxicity of TCDD between two rat strains, sensitive Long-Evans (Turku/AB) (L-E) and resistant Han/Wistar (Kuopio) (H/W), but there is another, currently unknown contributing factor involved. In the present study, we examined AHRR structure and expression in these rat strains to find out whether AHRR could be this auxiliary factor. Molecular cloning of AHRR coding region showed that consistent with AHRR proteins in other species, the N-terminal end of rat AHRR is highly conserved, but PAS B and Q-rich domains are severely truncated or lacking. Identical structures were recorded in both strains. Next, the time-, dose-, and tissue-dependent expression of AHRR was determined using quantitative real-time RT-PCR. In liver, AHRR expression was very low in untreated rats, but it increased rapidly after TCDD exposure (100microg/kg). Testis exhibited the highest constitutive expression of AHRR, whereas kidney, spleen, and heart showed the highest induction of AHRR in response to TCDD treatment. Again, no marked differences were found between H/W and L-E rats, implying that AHRR is not the auxiliary contributing factor to the strain difference in TCDD sensitivity. However, simultaneous measurement of CYP1A1 mRNA reinforced the view that AHRR is an important determinant of tissue-specific responsiveness to TCDD.     

Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin produces alterations in cortical neuron development and a long-term dysfunction of glutamate transmission in rat cerebral cortex

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is considered one of the most toxic dioxin-like compounds. It is ubiquitous in foodstuffs of animal origin and accumulates in the fatty tissues of animals and humans. Prenatal TCDD exposure has been associated, beside other effects, with persistent impaired cognitive development. In the present study, the effects of maternal exposure to TCDD during pregnancy on cortical neuron development at birth and cortical glutamate transmission in new-born, 14- and 60-day-old rat offspring, were investigated. A single dose (0.7μg/kg) of TCDD dissolved in corn oil was orally administrated to the dams on gestational day 18; controls dams were treated with the vehicle. All the experiments have been performed on the male offspring from vehicle-treated (i.e. control group) and TCDD-treated dams. Primary cultures of cerebral cortical neurons obtained from 1-day-old rats born from mothers exposed to TCDD displayed a reduction in cell viability (MTT assay) and an increase in the number of apoptotic nuclei (nuclear staining with Hoechst 33258) possibly associated with altered dendrite outgrowth (MAP2-immunoreactivity) with respect to control cell cultures. These changes were associated with impairment in cortical glutamate transmission, characterized by a reduction in basal and K(+)-evoked outflow as well as a decrease in [(3)H]glutamate uptake. Interestingly, the prenatal TCDD-induced alteration of cortical glutamate signaling is persistent since it was also present in 14- and 60-day-old offspring. Taken together, these results suggest that a single prenatal exposure to TCDD produces alterations in cortical neuron development associated with a long-term dysfunction of glutamate transmission in rat cerebral cortex. The possible relevance of these findings for the understanding of the long-lasting cognitive deficit observed in the offspring from mothers exposed to the toxicant during pregnancy, is discussed.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes.     

The role of aryl hydrocarbon receptor and the reactive oxygen species in the modulation of glutathione transferase by heavy metals in murine hepatoma cell lines

Glutathione transferase (GST) is a phase II detoxifying enzyme that plays a protective mechanism against oxidizing substances and toxic contaminants. Among these contaminants, heavy metals and polycyclic and halogenated aromatic hydrocarbons (PHAHs) have been shown to exert their toxic effects through the modulation of detoxifying enzymes, including the GSTs. Recently, we showed that heavy metals particularly Hg2+, Pb2+, and Cu2+ modulate the expression of phase II detoxifying enzymes such as NAD(P)H:quinone oxidoreductase 1 and Gsta1 in a concentration- and time-dependent manner. However, the effect of heavy metals and their potential interactions with aryl hydrocarbon receptor (AhR) ligands, PHAHs, on total Gst activity is still unknown. In the current study, we have investigated the effects of Hg2+, Pb2+, and Cu2+ in the absence and presence of four AhR ligands on the total Gst activity and reactive oxygen species (ROS) production in wild-type and AhR-deficient Hepa 1c1c7 cells. Our results showed that Hg2+ and Cu2+, but not Pb2+, significantly induced Gst activity in wild-type cells, whereas all metals induced the Gst activity in AhR-deficient cells. The induction of Gst activity by heavy metals was strongly correlated with an increase in the ROS production in wild-type, but not in AhR-deficient cells. Co-administration of heavy metals with AhR ligands differentially modulated Gst activity, in that co-exposure to Hg2+ plus AhR ligands could be beneficial in protecting against cytotoxicity as demonstrated by the increase in Gst activity with a proportional decrease in ROS production. Whereas co-exposure to Cu2+ plus AhR ligands was more toxic in that a decrease in Gst activity and an increase in oxidative stress of the cell were observed. We concluded that heavy metals differentially modulate the Gst activity through oxidative stress- and AhR-mediated mechanisms.