CYP1A1 is one of the most highly induced genes in the liver and extrahepatic tissues of experimental animal models following AHR activation; however, its contribution to toxic end points other than cancer has been investigated in only a few instances. CYP1A1 KO mice are resistant to overt toxicity and mortality induced by a single high dose of TCDD, but no cardiovascular end points were investigated (Uno et al., 2004
). Although CYP1A antisense morpholinos protected against TCDD-induced vascular dysfunction in zebrafish embryos in one study (Teraoka et al., 2003
), they were not protective in another (Carney et al., 2004
). Thus, our results establish for the first time that induction of CYP1A1 is an essential mediator of cardiovascular toxicity resulting from chronic dietary TCDD exposure.
The mechanism by which CYP1A1 mediates this cardiovascular toxicity remains to be elucidated; however, induction of CYP1A1 in the endothelium of both conduit and resistance vessels suggests that the vascular endothelium may be a target. Other studies have shown that CYP1A1 is highly induced in vascular endothelium (Garrick et al., 2005
; Guiney et al., 1997
; Schlezinger and Stegeman, 2000
; Smolowitz et al., 1991
; Stegeman et al., 1991
) and that exposure to TCDD or other TCDD-like HAHs significantly impacts vascular structure and function (Jokinen et al., 2003
; Toborek et al., 1995
). The idea that the vascular endothelium is a target is further supported by our observations that TCDD induces endothelial dysfunction and that CYP1A1 induction is required to mediate this effect. It is notable that physiological levels of vascular shear stress induce endothelial expression of CYP1A1 in a manner consistent with an anti-atherogenic phenotype (Conway et al., 2009
). Thus, although physiological mechanisms that induce endothelial CYP1A1 may be vasculoprotective, our data demonstrate that sustained xenobiotic-mediated induction is injurious.
One mechanism that could link sustained CYP1A1 induction to endothelial dysfunction is the production of superoxide anion. Superoxide anion can inactivate the vasodilator, nitric oxide, producing peroxynitrite and reducing endothelial-dependent vasodilation, and in our experiments, the endothelial dysfunction was normalized by the superoxide dismutase mimetic and antioxidant, tempol. Our results show that CYP1A1 induction is required to mediate TCDD-induced increases in superoxide anion in cardiovascular tissues, including aorta, which is consistent with our previous observations that CYP1A1 is required for TCDD-induced increases in superoxide anion in aortic endothelial cells in culture (Kopf and Walker, 2010
). CYP1A1 itself could be the source of the superoxide anion via NADPH-dependent enzymatic uncoupling as has been shown in other models (Schlezinger et al., 2006
; Shertzer et al., 2004
; Zangar et al., 2004
). It is also possible that CYP1A1 induction leads to the production of arachidonic acid hydroperoxides, which can lead to the subsequent release of superoxide anion or that the hypertension itself leads to increased vascular ROS. Thus, the specific cause-and-effect mechanism by which CYP1A1 mediates TCDD-induced superoxide anion remains to be determined.
It is interesting that although TCDD-induced superoxide anion production in the aorta, heart, and kidney was CYP1A1 dependent, TCDD induction of H2
in the aorta was not. Because this is not consistent with our observations in cultured endothelial cells (Kopf and Walker, 2010
), it suggests that the source of H2
in the aorta is not CYP1A1 and may not be from the endothelium. It has been shown that AHR agonists can also induce gene targets within mouse aortic vascular smooth muscle cells (Karyala et al., 2004
) and TCDD-induced changes in gene expression can differ significantly between intact aorta and cultured cells (Puga et al., 2004
). Thus, although unexpected, it is not unreasonable that the source of TCDD-induced H2
in the aorta in vivo
is not CYP1A1 and it is plausible that the source is not from the endothelium. It is possible that the enhanced CYP1B1 expression in the TCDD-exposed CYP1A1 KO mice could contribute to the increase in H2
. The tissue in which we observed the increase in CYP1B1 mRNA expression (heart) does not correspond to tissue in which we measured an increase in H2
(aorta). Nonetheless, differences in CYP1B1 protein expression as well as the antioxidant capacity of the respective tissues could account for this apparent discrepancy. Future studies will be needed to delineate the specific sources of cardiovascular ROS and the mechanisms by which CYP1A1 mediates the increase in superoxide anion.
In addition to being required to mediate TCDD-induced superoxide anion and endothelial dysfunction, CYP1A1 was also required for TCDD-induced hypertension. Increased ROS and vascular dysfunction are causally associated with hypertension in experimental animal models, particularly those mediated by activation of the RAS (Didion et al., 2002
). Although our data suggest that TCDD does not activate the RAS, we cannot completely rule out the possibility that there is an increased activation of angiotensin II receptors. Future studies using angiotensin II receptor blockers and antioxidant therapy and evaluating vascular reactivity of resistance vessels are needed to confirm the degree to which ROS, RAS, and vascular dysfunction contribute to TCDD-induced hypertension.
Lastly, although CYP1A1 KO mice were resistant to TCDD-induced cardiovascular toxicity, they exhibited phenotypic characteristics that were distinct from WT mice. CYP1A1 KO mice were significantly smaller than their age-matched WT controls, and MAP of CYP1A1 KO mice was slightly elevated (+8 mmHg). Although there was no evidence that this mild increase in blood pressure resulted in hypertension-related organ damage, such as cardiac hypertrophy, cardiovascular ROS, or vascular dysfunction, the increased MAP could contribute to an impairment of growth rate. The other notable difference between the CYP1A1 WT and KO mice was that TCDD-treated CYP1A1 KO mice exhibited an enhanced response to ACh-mediated vasocontraction at low doses. This suggests that TCDD may increase an endothelium-dependent contracting factor in CYP1A1 KO mice. One potential candidate would be cyclooxygenase 2, which is inducible by AHR activation (Kraemer et al., 1996
) and which can increase the metabolism of arachidonic acid to prostanoids that mediate vasocontraction. Future studies are needed to determine whether cyclooxygenase 2 plays a role in TCDD-induced vascular dysfunction.
As noted earlier, epidemiology studies have linked exposure to TCDD and TCDD-like HAHs to hypertension in humans and these associations have been noted for both high exposure levels as well as to current background exposure levels (Everett et al., 2008a
; Kang et al., 2006
; Uemura et al., 2009
). To compare our data with human exposures, we followed a similar dosing protocol (5 days/week for 35 days) as reported by Diliberto et al. (2001)
and used the measured liver concentration (6–7 ng/g) as an index of total body burden. Thus, we estimated that the final body burden of these mice was 600–700 ng/kg body weight. Although this body burden is 15–100 times higher than current exposure levels to TCDD and TCDD-like HAHs in the United States (Ferriby et al., 2007
), it is within the range estimated for Vietnam veterans and other individuals with known accidental or occupational dioxin exposure (100–8000 ng/kg body weight) (DeVito et al., 1995
; Emond et al., 2005
). Thus, our study provides biological plausibility for the link between human hypertension and exposure to TCDD-like chemicals. Nonetheless, future studies will be needed to establish the sensitivity of the hypertension response to TCDD and the degree to which even low level exposure may increase the susceptibility to hypertension in the presence of other common risk factors.
In summary, our study provides experimental evidence that induction of CYP1A1 is a risk factor for vascular dysfunction and hypertension. Human exposure to AHR agonists that induce CYP1A1 is not limited to TCDD-like HAHs but also includes the polycyclic aromatic hydrocarbons found in tobacco smoke, particulate matter air pollution, and the diet. Thus, induction of CYP1A1 could represent an important risk factor for cardiovascular disease for a large number of individuals as a result of environmental pollutant exposure.