As a consequence of organism utilization of oxygen, deleterious reactive oxygen species are produced. These oxygen species may lead to lipid peroxidation, DNA strand breaks and cell death. However, several antioxidant systems, such as GSH, SOD, catalase and MT have evolved to protect the organism from oxidative stress. A wide variety of stresses, ranging from physical injury to oxidative stress, induce MT in animals [27, 34]. Although numerous studies have been performed to confirm MTs antioxidative function, few have focused on the link between regulation of MT and a role during oxidative stress. In the present study we aimed to characterize the regulatory role of the rtMT-A enhancer region in response to oxidative stress and tumor promotion. Previous identification and functional analysis of distal elements on the rtMT-A promoter have revealed that a cluster including 4 AP1 elements and a single NF-IL-6 element may be involved in free radical inducibility . While free radical regulation of mammalian MT genes seem to be mediated via USF/ARE and MRE cis-acting elements , teleost MT genes may be regulated via conserved clusters of cis-acting elements sharing high homology to the NF-IL6 and the AP1 consensus core sequences [7, 11, 12]. Functional analysis, from the present study, of the rtMT-A promoter suggest that the NF-IL-6 element is instrumental to MT induction by oxidative stress (PQ), and the AP-1 elements may to a minor but significant extent contribute to free radical inducibility.
The observed absence of ROS induced MRE activation in the present study demonstrates that MT regulation in rainbow trout differ from that in mammals where the MRE binding transcription factor MTF-1 is activated following oxidative stress . Since the MRE elements identified on the rtMT-A promoter were not observed to contribute to the oxidative response this suggests that teleost MTF-1 is not activated by oxidative stress. Characterization of MTF-1, from zebrafish and rainbow trout has revealed a high conservation with regard to binding specificity and properties . However, this study points out that there might be different mechanisms that regulate MT gene expression during oxidative stress in different species.
While USF/ARE and MTF-1 mediate oxidative MT expression in mammals, the AP1 cis-acting elements identified on the rtMTA promoter, sharing high homology to the ARE core sequence, showed weak activation in response to oxidative stress. The AP1 cis-acting element was originally discovered on the hMTIIa gene mediating optimal basal level expression of MT . Functional analysis of the identified rtMT-A elements strongly indicated that the AP1 elements were required for maximal basal level expression in both fish (RTH-149) and mammalian cell (Hep G2) systems. Further analysis of binding affinity for the AP1 consensus sequence indicate that the proximal AP1 pair exhibited highest binding affinity, while the binding activity of the distal AP1 elements was at the border of detection limit. In addition, mutational analysis indicated that AP12 showed highest binding of the proximally located pair. Hence, functional analysis of the identified AP1 elements suggests that AP11,2 is functional with respect to both AP1 protein complex interaction and transactivation. However, there have been conflicting reports on the involvement of AP1 in the free radical regulation of MT in teleosts [30, 31]. While a deletion construct containing MRE and AP1 responded to ROS in common carp , the zebrafish MT gene promoter that contains both AP1 and MRE elements did not respond to ROS [30, 31]. In the present study co-transfection of Hep G2 cells with rtMT-A AP1 containing constructs with rtMT-A AP1 oligonucleotides abolished H2O2 induced promoter activity. These data suggest that the identified AP1 elements on the rtMT-A promoter in rainbow trout specifically mediate free radical MT inducibility. The primary role of the AP1 protein complex is as a modulator of cell proliferation and differentiation. It has been indicated that there is a link between proliferating human hepatic cells and high expression of MT protein . Exposure of RTH-149 cells to the tumor promoter PMA resulted in a strong activation of AP1 cis-elements in the present study. In vivo injection of rainbow trout with PMA strongly induced hepatic rtMT-A mRNA levels, confirming the functionality of the AP1 elements on the rtMT-A promoter. These data strengthen the view of MT as a modulator of cell proliferation and differentiation. In the teleost CHSE-214 cell line MT becomes progressively methylated during prolonged subculturing, coinciding with a decrease in MT expression and reduced cell proliferation . It has been suggested that antioxidants such as ascorbate, α-tocopherol and β-carotene are inhibitory to differentiation . MT has also been suggested to alter the cellular redox state , indicating a role for MT during development and differentiation.
Cellular responses following stress, such as injury, promote a transient activation of NF-IL6 and AP1 protein that both play key roles in the initiation of inflammatory responses. NF-IL6 is rapidly activated in response to cytokines and oxidative stress . This is in support with the present study in the RTH-149 cell line demonstrating that the NF-IL6 element was substantially activated in response to PQ but not to PMA. NF-IL6 sites are also present in the MT enhancer of other teleosts such as the crucian carp  suggesting a conserved function of MT in response to inflammation where NF-IL6 may mediate ROS inducibility. Studies have demonstrated that NF-IL6 activity can be modulated following protein-protein interaction with the AP1 protein complex  and NF-κB . The observed minor increase in AP1 element activity following PQ exposure may enhance MT expression to protect from oxidative stress in fish. Rainbow trout cells respond to ROS exposure by induction of MT, which result in cellular protection from ROS toxicity [3, 4, 6]. Furthermore, it has been shown in a recent study on cod that there is a close correlation between the hepatic levels of ROS and MT . IL6 and CXCL8 are two of the first inflammatory mediators expressed and contain cis-acting sites for both AP1 and NF-IL6, which indicate their crucial role in acute phase responses [44, 45]. Kanekiyo and colleagues  demonstrated that MT gene activation by zinc regulates macrophage colony stimulating factor (M-CSF), which in turn recruit and stimulate cytokine production by monocytes. Mice with genetic deletions in the MT proteins have a significant reduction in inflammatory mediators, including TNFα, IL6 and IL1α, compared to wild type mice . Furthermore, growth hormone (GH) was found to induce the expression of rtMT-A) . GH treatment results in phosphorylation of NF-IL6 and increases its transcriptional activity. This suggests that GH may be able to modulate MT regulation through NF-IL6 signaling.