Department of Operations and Intelligence, Navy Environmental and Preventive Medicine Unit No. 2, Norfolk, Virginia
Received January 30, 2004; accepted January 30, 2004
The U.S. Environmental Protection Agency reassessment of the health effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related compounds in part re-evaluates approaches to low-dose extrapolation of risk for these compounds (U.S. EPA, 2000). While a multitude of models describing the low-dose effects of TCDD have been presented over the past decade, an understanding of some of the biological factors defining the shape of the dose-response curve at low doses has become increasingly detailed only in the last few years, with the application of quantitatively sensitive technical approaches. Clearly, understanding the early events leading to molecular and cellular responses to TCDD exposure is crucial for low-dose extrapolation of risk.
The mechanistic basis of action of TCDD and related compounds is one of the most extensively studied in toxicology, and a hallmark of exposure to these compounds in experimental systems is induction of the cytochrome P4501A and 1B (CYP1A, CYP1B) family of enzymes. CYP1A1 induction in experimental systems is one of the most frequently cited markers of exposure to TCDD and related compounds because of its low level of constitutive expression and robust increase in response to aryl hydrocarbon receptor (AhR) ligand. The mechanism of CYP1A1 induction has been well characterized. However, a full understanding of the obligate molecular and cellular events that precede physiological and toxic responses to AhR ligands has not hitherto been described.
TCDD-induced CYP1A1 protein expression in intact rat liver following in vivo treatment is regiospecific and dose-dependent, with expression localized within the centrilobular region at low doses and induction in the periportal regions increasing with dose (Tritscher et al., 1992). At any given dose, a clear boundary between induced and uninduced regions of intact liver can be observed by immunohistochemistry, and this heterogeneous pattern of induction suggests that a binary switching mechanism may govern expression of CYP1A1 in rat liver (Bars and Elcombe, 1991). Andersen and colleagues developed several physiologically based pharmacokinetic (PBPK) models that capture responses of hepatocytes within regions as well as the average response of the intact liver, but these models lacked the mechanistic detail that would account for the observed differential sensitivity in individual cells (Andersen et al., 1997a
,b
). Recent work, including the two highlighted articles in this issue of Toxicological Sciences, examines CYP1A1 induction patterns in individual cells and in cell populations, and it lays the groundwork for further investigation into control mechanisms regulating the early stages of CYP1A1 gene and protein expression.
Using primary rat hepatocytes, French et al. (2004) examine induction of CYP1A1 gene expression in individual cells and in populations of cells exposed to the potent AhR agonist, 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB 126). Observations in individual hepatocytes using immunocytochemistry (ICC) and in situ hybridization (ISH) revealed an "all-or-none" or binary response to a 24-h exposure to a six-fold concentration range of PCB 126. For both protein and mRNA endpoints, the authors observe an increase in the number of induced cells with increasing concentration, with some hepatocytes remaining nonresponsive to stimulus, even at the highest concentration tested. Population responses showed time- and concentration-related increases in induction of CYP1A1 mRNA and protein; these observations are consistent with a multitude of reports in the literature. Taken together, individual cell- and population-induction responses do not follow a two-population distribution, with one population of cells in the basal state and one fully induced population. The data do reveal a basal population and a time- and concentration-dependent, variably induced population. These observations support the conclusion that a "hybrid" switch response may be at work, with a binary switching mechanism responsible for whether a gene is active or not and a second, rheostat-type switch governing variability in transcriptional output.
In a second paper in this issue of Toxicological Sciences, Broccardo and colleagues (2004) extend this primary rat hepatocyte model, focusing on the H4IIE and Hepa1c1c7 rodent hepatoma cell lines. Using similar technical approaches, the authors compare PCB 126-induced CYP1A1 mRNA and protein expression, both in individual cells and populations of cells. Flow cytometry and ICC experiments show that populations of cells display a continuous dose-response curve for both CYP1A1 mRNA and protein induction, moving from an uninduced to an induced state in a binary fashion. Individual cells display varying dose-dependent degrees of induction and, in some cells, even the highest concentrations tested in this series of experiments did not induce CYP1A1 gene expression. Some noteworthy differences were observed between the two cell lines. Specifically, the H4IIE cells were more sensitive than the Hepa1c1c7 cells to PCB 126 exposure, leading the authors to conclude that the switch behavior was more pronounced in the H4IIE cells. Results from studies in both cell lines support the hypothesis of a hybrid switch process governing CYP1A1 expression in rodent liver cell lines.
Regulation of CYP1A1 and other TCDD-inducible genes relies on a multitude of factors. In the accepted CYP1A1 induction model, TCDD and related compounds bind specifically to the AhR, inducing conformational changes that affect interaction with the Arnt cofactor and basal transcriptional machinery, ultimately forming a complex that regulates transcription through dioxin-responsive elements (DREs). This model is relatively straightforward, primarily involving proteinprotein and proteinDNA interactions and other spatial relationships, with each required step in the induction process acting as a potential switch regulating gene induction.
With rare exception in eukaryotic systems, genes are either maximally expressed or not expressed at all at the individual cell level. Binary control is attributable, at least in part, to the inherent characteristics of gene networks, including competitive binding of transcriptional activators and repressors to multiple binding sites within a promoter and cooperative and synergistic interactions between transcription factors and promoter elements (Louis and Becskei, 2002). Other genomic events, such as chromatin remodeling, present other potential sources of binary switching (Okino and Whitlock, 1995
). However, graded and binary modes of transcriptional regulation are not necessarily intrinsic only to gene networks, but also rely on other potentially complex regulatory arrays, including nongenomic controls (Biggar and Crabtree, 2001
).
The mitogen-activated protein (MAP) kinase cascade is an example of a nongenomic control mechanism facilitating spatially distinct events working in concert to rapidly activate a signaling cascade. At near saturation concentrations, the MAP kinase system can convert continuous inputs into binary outputs through a kinase cascade mechanism leading to ultrasensitivity (Huang and Ferrell, 1996). Tan and colleagues have demonstrated that the MAP kinases ERK and JNK are activated independently of the AhR yet are essential for induction of CYP1A1 gene transcription via the AhR (Tan et al., 2001). TCDD-like compounds have been shown to activate protein kinase C and phosphorylation states of the AhR and Arnt proteins are critical features of the CYP1A1 induction process (Chen and Tukey, 1996
; Hanneman et al., 1996
; Long et al., 1999
; Park et al., 2000
). As such, phosphorylation/dephosphorylation processes present yet another element with switch-like potential in this increasingly complex regulatory system.
A ligand-activated transcription factor, the AhR is structurally distinct but functionally similar to members of the steroid hormone receptor family. Nongenomic responses to steroid exposure have been described extensively for steroid hormone signaling, shifting the view that this signaling system is exclusively mediated by nuclear receptor function (Cato et al., 2002). Such dynamic regulatory models incorporate both ligand- and protein kinase-signaling mechanisms and also consider the important role of protein stability in addition to transcriptional function. Protein stability and other potential nongenomic actions that may regulate transduction of dioxin-like signals through a hybrid switch mechanism include effects on intracellular communication and other nonreceptor-mediated effects at the plasma membrane, effects mediated through a hitherto unidentified membrane-bound receptor, and effects that occur through membrane-localized action of the AhR.
Both highlighted papers reinforce the need to re-examine nonlinear, low-dose extrapolation of the dose-response curve for AhR agonists within the context of increasingly complex transcriptional regulation of CYP1A1 and other members of the Ah gene battery. Evaluating switching mechanisms will provide valuable information in risk assessment of this important class of environmental contaminants and could potentially identify novel targets in this signaling pathway that integrate nongenomic and genomic regulatory actions.
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1 To whom correspondence should be addressed at NEPMU-2, 1887 Powhatan St., Norfolk, VA 23511. Fax: (757) 444-1191. E-mail: wilsonc{at}nepmu2.med.navy.mil
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