Our results demonstrate that subchronic TCDD exposure increases systemic arterial blood pressure, left ventricle weight and wall thickness, cardiovascular superoxide, and induces endothelial dysfunction characterized by a reduction in NO-dependent vasorelaxation. This study represents the first animal model that definitively demonstrates that sustained AhR activation by TCDD induces hypertension and cardiac hypertrophy, associated with impaired vascular reactivity.
Systolic blood pressure above 130 mmHg is considered hypertensive in mice [22
]. Therefore, the blood pressure of 131/106 mmHg observed in TCDD-exposed mice at the termination of the study would classify them as hypertensive. This increase in blood pressure is consistent with previously reported increases in blood pressure from a mouse model of acute TCDD exposure as measured by a tail cuff system [8
]. However, we observe an initial increase in blood pressure as early as day 2–3, while Dalton et al. [8
] did not see significant increases in blood pressure until day 23–29. This may be due in part to the differences of dose and exposure, as well as the increased sensitivity that radiotelemetry provides for detecting smaller changes in blood pressure [23
]. Moreover, the telemetry recordings allowed us to observe that the TCDD-exposed mice developed a nondipping pattern of diurnal blood pressure, characterized by less than 10% differences in daytime and nighttime blood pressure. Hypertensive target organ damage is more prevalent in individuals with a nondipper blood pressure pattern [24
], suggesting that TCDD-induced hypertension may lead to significant organ damage as observed with the left ventricle hypertrophy. Furthermore, the increase in LV + S weight, but not in RV weight, in TCDD-treated mice is indicative of an increase in systemic arterial pressure in the absence of an increase in pulmonary arterial pressure.
The TCDD-induced hypertension is also associated with a significant impairment of NO-dependent vasodilation that is normalized by a superoxide dismutase mimetic tempol. Superoxide is frequently elevated in human and experimental hypertension [11
] and an important consequence of superoxide production is the loss of endothelium-derived NO. NO is as a potent vasodilator and genetic deletion of the enzyme that produces it, endothelial nitric oxide synthase (eNOS), results in loss of endothelial-dependent vasodilation (i.e., endothelial dysfunction) and hypertension [26
]. Increased vascular superoxide reacts with NO to produce peroxynitrite and thus represents a key mechanism by which NO bioavailability is reduced, leading to diminished endothelial-dependent vasorelaxation. Furthermore, treatment with antioxidants, including tempol, improves vascular function and attenuates hypertension in many animal models [27
], similar to our results in isolated artery studies. Moreover, chronic TCDD exposure increases the severity of atherosclerotic plaques in apolipoprotein E null mice [8
], which may be attributed to a decrease in bioavailable NO and its anti-atherosclerotic activity [18
]. Nonetheless, we cannot rule out the possibility that TCDD exposure reduces NO-dependent signaling in the vascular smooth muscle. Future studies of the aortic vasorelaxation responses to NO donors would establish whether downstream NO signaling is also altered by TCDD.
It has been well established that TCDD and other AhR ligands induce oxidative stress in humans and experimental animal models. TCDD exposure has been shown to induce oxidative stress in brain [28
], liver [28
]), and kidney tissue [12
] of mice. In rats, oxidative stress is increased in brain [30
], liver [30
], and reproductive tissue [30
] by exposure to TCDD, polychlorinated dibenzofurans, or polychlorinated biphenyls. In humans, serum polychlorinated dibenzo-p
-dioxin and polychlorinated dibenzofuran levels and plasma lipid peroxidation were positively correlated in metal recovery workers [31
]. Moreover, concomitant antioxidant supplementation has been shown be protective in TCDD-induced toxicity in mice [32
In the cardiovascular system, 3,3′,4,4′-tetrachlorobi-phenyl (PCB 77), a TCDD-like AhR agonist, has been shown to increase ROS in endothelial cells [33
]. Moreover, exposure of zebrafish embryos to TCDD results in decreased blood flow in the mesencephalic vein, which is abolished by simultaneous exposure to an antioxidant [34
]. Additionally, occupational exposure of pesticide production workers to TCDD is associated with impaired microvascular reactivity, which was negatively correlated with superoxide dismutase activity [35
], indicative of vascular superoxide and endothelial dysfunction. Thus, activation of AhR appears to increase ROS in multiple organ systems.
There are a number of potential mechanisms for these observed increases in ROS. First, antioxidant enzyme activity could be suppressed, increasing susceptibility to endogenous ROS. TCDD exposure has been shown to decrease catalase and glutathione peroxidase activity in rat brain tissue [36
] and decrease glutathione peroxidase, glutathione reductase, and SOD activity in chicken liver [37
]. Since we see modest increases, rather than decreases, in mRNA expression of antioxidant response genes, these data suggest that suppressed antioxidant responses are not the primary cause of the increased ROS.
A second potential mechanism is increased mitochondrial production of ROS. Mitochondria are responsible for the majority of ROS production in most cells. However, ROS production and elimination by antioxidant systems is balanced under normal conditions [38
]. Mitochondrial dysfunction that elevates ROS release can lead to the development of cardiovascular disease, particularly atherosclerosis and hypertension [39
], and AhR-dependent mitochondrial production of ROS has been shown to be increased following TCDD exposure in mouse liver [40
]. Interestingly, we saw an increase in mRNA expression of the mitochondrial isoform of SOD (SOD2 or MnSOD) in the left ventricle of TCDD-exposed mice. These data suggest that the source of the ROS could be mitochondrial, potentially linked to the mitochondrial accumulation of CYP450s that follow TCDD exposure [41
Uncoupling of eNOS is a third potential source of ROS, where the enzyme transfers an electron from NADPH to oxygen, rather than the substrate, L-arginine, resulting in the formation of superoxide instead of NO [42
]. The primary mechanism by which eNOS uncoupling occurs is from a decrease in the availability of the co-factor tetra-hydrobiopterin (BH4
levels can be reduced as a result of impaired synthesis and/or increased oxidation, both of which can lead to eNOS uncoupling, endothelial dysfunction, and hypertension [44
]. Microarray studies have shown that TCDD exposure decreases the mRNA expression of cyclohydrolase 1 (GTPCH), the rate limiting step in BH4
synthesis, and increases the mRNA expression of GTP cyclohydrolase negative feedback protein, which inhibits BH4
]. Thus, sustained AhR activation by TCDD may lead to BH4
depletion, eNOS uncoupling, and increased vascular superoxide.
Lastly, uncoupling of the induced cytochrome P450s is a fourth potential source of ROS. It has been demonstrated that P450-enriched human microsomes are capable of producing ROS [49
]. Moreover, TCDD-induced mouse liver microsomes have increased production of superoxide [50
]. CYP1A1 is of particular interest as a potential source of ROS. It is dramatically induced in the tissues of TCDD-exposed mice in our study, including the vasculature, and CYP1A and CYP1A1 are inducible in endothelial cells by AhR activation [51
]. Furthermore, CYP1A and CYP1A1 have been shown to contribute to TCDD-induced toxicity in zebrafish and mice, respectively [52
]. Future studies will investigate the role of CYP1A1 in ROS production and cardiovascular toxicity.
Hypertension in both humans and animal models is often associated with activation of the sympathetic nervous system and/or elevation of circulating Ang II and ET-1 [53
]. Heart rate was not significantly altered in TCDD-exposed mice, suggesting that sympathetic nervous system activity was not increased. Similarly, neither plasma Ang II nor ET-1 was significantly altered in TCDD-exposed mice. While these data do not provide evidence of the involvement of these pathways, our results also do not rule them out. For example, increased sympathetic tone of peripheral resistance vessels can increase blood pressure without increasing heart rate [53
], while plasma ET-1 levels do not necessarily reflect the amount of vasoactive ET-1 [54
]. Therefore, future studies using pharmacological inhibitors of these pathways will help to further define their contribution to TCDD-induced hypertension.
While the TCDD exposure resulted in hypertension, it also induced periodic normalizations of the blood pressure. The mechanism underlying these normalizations in blood pressure is unknown, but may include a compensatory decrease in autonomic nervous system activity, changes in the tissue distribution of TCDD over time, or pressure natriuresis, an increase in sodium and water renal excretion in response to an increase in renal arterial pressure. A decrease in autonomic activity commonly is an acute compensatory response occurring over seconds to minutes rather than days, while significant changes in TCDD tissue distribution would not be expected to occur during these periods of blood pressure normalizations, based on previous reports of the toxicokinetics of TCDD in the mouse [17
]. Pressure natriuresis is a possible explanation, which could be verified by determining if urine volume and sodium excretion increase during the normalization periods. Moreover, if pressure natriuresis is responsible for the normalizations, the observed hypertension would be salt sensitive. Future studies of the mechanisms responsible for TCDD-induced increases in blood pressure will likely help to elucidate the reasons for the periodic normalizations.
In conclusion, our model is the first to definitively demonstrate that sustained AhR activation by subchronic TCDD exposure induces hypertension, vascular dysfunction, and cardiac hypertrophy. These data are consistent with the epidemiology studies of Vietnam veterans exposed to Agent Orange and validate the need to continue epidemiology studies of such cohorts as they reach the age when cardiovascular morbidity and mortality increase further. This model should provide valuable insight into the mechanisms underlying TCDD-induced cardiovascular pathogenesis, including the role of vascular ROS as potential mediators of TCDD-induced hypertension.