Ferritin, by regulating the "free" intracellular iron pool, controls iron-catalyzed generation of reactive oxygen species, but its role in oxidative damage is still unclear. We show that ferritin synthesis is significantly stimulated in the liver of rats subjected to oxidative stress by treatment with phorone, a glutathione-depleting drug. RNA-bandshift assays document reduced activity of iron regulatory factor, in particular of IRFB, the cytoplasmic protein that post-transcriptionally controls ferritin mRNA translation. Furthermore, Northern blot analysis shows increased accumulation of H and L subunit mRNAs, and nuclear run-on experiments provide evidence of transcriptional activation. Direct measurements of intracellular free iron levels by EPR indicate that the increased ferritin synthesis can be mediated by an expansion of the free iron pool. An early drop of ferritin content after phorone treatment indicates that part of the iron that fuels the free pool might derive from ferritin degradation. Present data seem to suggest that, under conditions of oxidative stress, liver ferritin can represent either a pro- or an anti-oxidant in a time-dependent manner. In fact, its early degradation contributes to expand the intracellular free iron pool that, later on, activates multiple molecular mechanisms to reconstitute ferritin content, thus limiting the pro-oxidant challenge of iron.

Post-ischaemic reperfusion increases the level of the major heat-shock (stress) protein hsp 70 and of its mRNA by transcriptional mechanisms, and activates the binding of the heat-shock factor HSF to the consensus sequence HSE. In common with CoCl2 treatment, post-ischaemic reperfusion increases the level of haem oxygenase mRNA, an indicator of oxidative stress, but CoCl2 does not seem to induce the expression of the hsp 70 gene [Tacchini, Schiaffonati, Pappalardo, Gatti and Bernelli-Zazzera (1993) Lab. Invest. 68, 465-471]. Starting from these observations, we have now studied the expression of two genes of the hsp 70 family and of other possibly related genes under conditions of oxidative stress. Three different chemicals, which cause oxidative stress by various mechanisms and induce haem oxygenase, enhance the expression of the cognate hsc 73 gene, but do not activate the inducible hsp 70 gene. Expression of the other genes that have been studied seems to vary in intensity and/or time course, in relation to the particular mechanism of action of any single agent. The pattern of induction of the early-immediate response genes c-fos and c-jun observed during oxidative stress differs from that found in post-ischaemic reperfused livers. Oxidative-stress-inducing agents do not promote the binding of HSF to its consensus sequence HSE, such as occurs in heat-shock and post-ischaemic reperfusion, and fail to activate AP-1 (activator protein 1). With the possible exception of Phorone, the oxidative stress chemically induced in rat liver activates NFkB (nuclear factor kB) and AP-2 (activator protein 2) transcription factors.

No abstract

induction of transcription of the hsp 70 gene and the increase The aim of this study was to investigate the behavior of in the steady-state levels of hsp 70 messenger RNA. All these the transcription factors, heat-shock factor (HSF) and nuclear changes are primed by activation and binding of the specific factor kB (NF-kB), in postischemic reperfused liver, with partranscription factor, HSF (heat shock factor), to the correticular attention paid to possible differences in the time-course sponding consensus sequence, HSE (heat shock element), in and mechanism of activation, which may help in defining the promoter of heat shock genes. 1-3their role in the response of the liver to reperfusion. Ischemia Some other genes, such as the immediate early response was induced by clamping the hilar pedicle of the left lateral genes, c-fos and c-jun, are induced in postischemic reperfused and median liver lobes; the clamp was removed after 1 hour.liver; in particular, the concurrent induction of heme oxySome rats were treated intraperitoneally with IL-1 receptor genase 1 suggests the existence of a state of oxidative stress.3 antagonist (IL-1RA) 30 minutes before ischemia and at the The association of a state of oxidative stress with blood repertime of reperfusion. Binding of NF-kB to the corresponding fusion is a long-established fact, although recent observations consensus sequence is activated after 30 minutes of reperfuhave mainly emphasized the role of invading leukocytes in sion, and is still increased 1 hour after reperfusion. Activation the generation of a delayed, second wave of reactive oxygen is suppressed in rats treated with IL-1RA; NF-kB persists in species (ROS), particularly in the liver. [4][5][6][7] Oxidative stress the cytosol associated with the inhibitor, IkB, and can be occurring during postischemic reperfusion is superimposed artifactually activated in vitro. Super-gel shift experiments revealed that the two subunits, p50 and p65, are involved in on molecular changes in cells that have been subjected to the activation of binding. In contrast, binding of HSF to the ischemia. When ischemia occurs under conditions devised corresponding consensus sequence, heat shock element for the storage of livers destined for transplantation, the (HSE), is already activated at the end of ischemia, shows a events that occur at reperfusion differ from warm ischemia, further increase after 30 minutes of reperfusion, but declines realized by clamping blood vessels inside the body; in partic-1 hour after reperfusion; more importantly, it is not inhibited ular, reperfusion after cold ischemia does not induce hsp by pretreatment of the rat with IL-1RA. In conclusion, al-70.8 There are also distinct differences in the pattern of gene though both HSF and NF-kB are activated by ischemia-reper-expression between rat livers undergoing postischemic reperfusion, there are clear differences in time-course and mecha-fusion and those subjected to oxidative stress induced by nism of activation of the two transcription factors. Activation...

called ''heat shock proteins'' (hsps). [4][5][6][7][8][9] Not only the response to The expression of hsp70-the inducible member of the temperature is conserved but the heat-inducible proteins are corresponding heat shock gene family-of the oxidative conserved as well; among them, the hsp70 family of hsps are stress marker gene heme oxygenase (HOx), and of the particularly important for amount, constancy of induction, immediate early response genes c-fos and c-jun has been and possible role as cellular thermometer regulating the exstudied in FAO hepatocarcinoma cells depleted of polypression of all hsps. [10][11][12] Heat shock has additional effects on amines and exposed to heat shock. Depletion of polymessenger RNA (mRNA) stability and translational control, amines was obtained in short-term experiments (24-48 which contribute to the preferential expression of hsps. Behours) by the use of a-difluoromethylornithine (DFMO), cause of their rapid induction, most of the studies on hsps a classical inhibitor of ornithine decarboxylase (ODC), have emphasized this characteristic response to heat but or of the combination of the newly available inhibitors of ODC and S-adenosylmethionine decarboxylase, i.e., many hsps are also constitutively expressed. The main func-(2R,5R)-hept-6-yne-2,5-diamine (MAP) and 5-{[(Z)-4-tion of constitutive and inducible hsps is to regulate the foldaminobut-2-enyl]methylamino}-5-deoxyadeno-sine (Ab-ing of newly synthesized protein and to help in their refolding eAdo). Under our experimental conditions polyamine im-during or after cell injury. 8,9 balance was realized without appreciable growth arrestIn addition to exposure to heat, other noxious agents, conand impaired expression of growth-related genes. De-nected in some way with protein malfolding or denaturation, creases of putrescine and spermidine 48 hours after can induce the synthesis of hsps 13 and may also be relevant DFMO prevented the induction of hsp70 messenger RNA to liver cell pathology.14,15 Indeed, it has been recognized that (mRNA), whereas depletion of spermidine and spermine ''the heat-shock response is for hepatologists, too '' 16 and that obtained with MAP/AbeAdo decreased intensity and du-the application of hsps to clinical medicine is on the horiration of post-heat shock accumulation of hsp70 mRNA. zon. 17,18 The induction of hsp70 mRNA starts with the activaInductions of HOx, c-jun and c-fos were also inhibited. tion of the heat shock transcription factor (HSF), which binds Because MAP/AbeAdo caused also an intracellullar accu-to the consensus sequence heat shock element (HSE) present mulation of putrescine, we tested the effect of exogenous in the promoter of heat shock genes and transactivates the putrescine, which was found to stabilize the mRNAs for gene. In some circumstances, however, the binding is not hsp70 and c-jun. Hsp70 and HOx are thought to play a followed by the appearance of hsp70 mRNA, 19,20 which argues protective role, and the proteins of c-jun and c-fos consti-for the need of permissive conditio...