Our results demonstrate that TCDD exposure of endothelial cells induces CYP1A1 and CYP1B1, increases the production of ROS, and decreases ACh-stimulated NO production. Moreover, the ability of AhR siRNA to prevent the TCDD induction of ROS demonstrates that the AhR is required. Furthermore, the ability of CYP1A1 siRNA to significantly attenuate TCDD-induced CYP1A1 expression, CYP1A1/1B1 enzyme activity, and completely prevent the TCDD-induced ROS production suggests that CYP1A1 is a downstream mediator of this ROS.
We observed an induction of CYP1A1 and CYP1B1 mRNA, protein, and enzymatic activity in TCDD-exposed HAECs. Others have shown that CYP1A1 is expressed and inducible in vascular endothelial cells (Farin et al., 1994
;Stegeman et al., 1995
;Thirman et al., 1994
;Toborek et al., 1995
), while CYP1B1 is expressed and inducible in vascular smooth muscle cells (Kerzee and Ramos, 2001
;Zhao et al., 1998
). The results of these previous studies suggested that in the vasculature CYP1A1 is exclusively expressed in endothelial cells, while CYP1B1 is exclusively expressed in smooth muscle cells. However, a recent study has demonstrated both CYP1A1 and CYP1B1 are induced in HAECs, as observed in our study, but only CYP1A1 is induced in primary human umbilical vein endothelial cells (HUVECs) (Eskin et al., 2004
). Thus, these data, combined with our results, suggest that expression and the potential for induction of CYP1A1 and CYP1B1 in endothelial cells may be species- and vascular bed-dependent.
TCDD exposure resulted in an increase in ROS production, which was maximal at a concentration of 1 nM. Superoxide anion production was significantly increased at TCDD concentrations of 0.1 and 1 nM, but not at 10 nM. In contrast, the detection of H2
and hydroxyl radical by DCF was significantly increased at all TCDD concentrations. It is possible that at the highest concentration of TCDD (10 nM) antioxidants had been induced to dismutate the superoxide anion. One possible antioxidant mediating this effect is NAD(P)H:quinine oxidoreductase 1 (Nqo1). Nqo1 is an effective scavenger of superoxide anion (Siegel et al., 2004
) and is considered part of the mouse AhR gene battery (Tijet et al., 2006
;Yeager et al., 2009
). Further, we have shown that TCDD significantly induces Nqo1 mRNA in the heart of mice (Kopf et al., 2008
) and in HUVECs after 24 h exposure (unpublished data).
The TCDD-induced increase in superoxide anion was paralleled by a concentration-dependent decrease in ACh-stimulated NO production. A key mechanism by which NO is reduced by oxidative stress is the reaction between NO and superoxide anion, producing peroxynitrite. Thus, it is consistent that that the 10 nM concentration of TCDD that did not significantly increase superoxide anion production also did not significantly reduce NO production. Further the increase in ROS and decrease in NO by TCDD was prevented by siRNA targeting AhR. These data demonstrate that the increase in ROS following TCDD exposure is AhR-dependent.
One gene that is notably upregulated downstream of AhR activation in endothelial cells and is associated with increased ROS production is CYP1A1. Our data not only show that AhR siRNA prevents both TCDD-induced CYP1A1 expression and ROS production, but go further to establish a cause-and-effect relationship between them, since CYP1A1 siRNA reduces TCDD-induced CYP1A1 expression and prevents TCDD-induced ROS. We found that CYP1A1 siRNA reduced TCDD-induced CYP1A1 expression by 66%, completely prevented the TCDD-induced increase in ROS, and attenuated the TCDD-induced decrease in NO production by 60%. These results suggest that CYP1A1 is an upstream mediator of the increased production of ROS and the decreased production of NO. There is extensive evidence that induced levels of CYP1A1 are capable of producing ROS. Studies of enriched preparations of microsomes of specific human CYP450s reveal that CYP1A1 is prone to the production of superoxide anion and H2
(Puntarulo et al., 1998
). Furthermore, microsomes from the liver of TCDD-exposed mice produce a greater amount of superoxide, compared to control mice (Shertzer et al., 2004a
). Moreover, microsomes from the livers of TCDD-exposed CYP1A1 null mice demonstrate a decrease in the production of H2
(superoxide anion was not measured), compared to TCDD-exposed wild type mice, suggesting CYP1A1 may be the primary source of ROS in TCDD-induced microsomes (Shertzer et al., 2004b
A second gene that is also notably upregulated downstream of AhR activation is CYP1B1. However, in contrast to our results with CYP1A1, siRNA targeting CYP1B1 did not reduce TCDD-induced ROS production or prevent the TCDD-induced decrease in NO, suggesting that CYP1B1 is not a mediator of the ROS in these cells following TCDD exposure. It is possible, however, that any decrease in CYP1B1 production of ROS by CYP1B1 siRNA was masked by an increase in CYP1A1. While CYP1B1 siRNA did not alter the basal expression of CYP1A1 mRNA, it did significantly increase it in the TCDD treatment group. The degree to which this modest increase (1.5x) in CYP1A1 mRNA results in an increase in CYP1A1 protein expression is not known. Despite the increase in CYP1A1 mRNA, the overall impact of CYP1B1 siRNA was a decrease in enzymatic activity, suggesting that CYP1B1 siRNA has a suppressive effect on the total TCDD-induced CYP activity. Nonetheless, to fully answer this question the relative redox coupling efficiencies of CYP1A1 and CYP1B1 would need to be directly compared and the ability of CYP1B1 to produce ROS in the absence of CYP1A1 would need to be determined. Our future studies assessing TCDD-induced ROS production in CYP1A1 null mice will help to address this question. In addition, a recent study suggests that CYP1B1 may reduce the oxidative state of endothelial cells. Retinas of CYP1B1 knockout mice and retinal endothelial cells isolated from CYP1B1 knockout mice exhibit increases in oxidative stress as evidenced by 4-hydroxy-2-nonenal staining and DHE fluorescence, respectively (Tang et al., 2009
). Furthermore, treatment of retinal endothelial cells from CYP1B1 knockout mice with antioxidants restores their angiogenic capability. In our studies CYP1B1 siRNA did not reduce basal levels of ROS in control cells or attenuate the ROS production induced by TCDD. Thus, our data do not provide evidence that CYP1B1 has an inherent antioxidant, protective function in HAECs.
It is well established that superoxide anion and H2
are common by-products of the P450 catalytic cycle, resulting from inefficient coupling of NADPH consumption to substrate oxidation (Zangar et al., 2004
) and this may represent one potential mechanism by which ROS can be produced downstream of CYP1A1 induction. If the activated oxygen is released from the heme iron immediately after the addition of the first electron (Fe2+
complex), superoxide will be produced. If the activated oxygen is released from the heme iron after the addition of the second electron (Fe2+
OOH complex), H2
will be produced (Parkinson, 2001
). The degree of coupled NADPH consumption and substrate oxidation varies between CYP450 isoforms, but it is often less than 50% for eukaryotic CYP450s (Gorsky et al., 1984
;Gruenke et al., 1995
;Kuthan and Ullrich, 1982
;Tan et al., 1997
Another potential downstream mechanism by which induced CYP1A1 can result in increased production of ROS is by the production of quinones, molecules containing a diketone six-carbon ring. A quinone can then be reduced to a semiquinone radical by a one electron transfer from NADPH-cytochome P450 reductase, which results in redox cycling and the production of ROS. For example, CYP1A1 preferentially metabolizes estradiol into 2-hydroxylestradiol and 4-hydroxylestradiol, respectively (Lee et al., 2003
;Spink et al., 1994
), metabolites that can contribute to cytotoxicity and mutagenicity by the downstream formation of catechol estradiols and subsequent oxidation to semiquinone radicals (Samuni et al., 2003
). This potential mechanism is intriguing and deserves further investigation to determine whether endogenous metabolites of CYP1A1 may undergo this metabolic activation.
In conclusion, this study demonstrates that TCDD exposure results in an increase in ROS production in endothelial cells that is AhR-dependent and downstream of CYP1A1 induction. As others have shown that CYP1A1 contributes to ROS production in isolated liver microsomes (Puntarulo and Cederbaum, 1998
;Shertzer et al., 2004b
), our results suggest this also occurs in human vascular endothelial cells. Finally, this study provides insight into a potential role of CYP1A1 in TCDD-induced hypertension and endothelial dysfunction in vivo. Future studies will use this cell culture model to investigate the underlying mechanism of CYP1A1-dependent ROS production.