Manipulation of Metallothionein Expression in the Regenerating Rat Liver Using Antisense Oligonucleotides

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 246, 711–718 (1998)ARTICLE NO. RC988612

Manipulation of Metallothionein Expression in theRegenerating Rat Liver Using Antisense Oligonucleotides

Vikram Arora,* Patrick L. Iversen,*,† and Manuchair Ebadi*,1

*Department of Pharmacology, University of Nebraska College of Medicine, Omaha, Nebraska 68198-6260; and†AVI Biopharma, 4575 S. W. Research Way, Suite 200, Corvallis, Oregon 97333

Received April 9, 1998

tween 5 and 7 g atoms of a group IIb heavy metalsMetallothioneins (MTs) are low molecular weight, such as zinc, copper, and cadmium per mole of protein.

zinc-binding proteins that by activating zinc metallo- The metal content of purified MT is highly variableenzymes participate in the regulation of growth and and depends on organism, tissue, and history of heavydevelopment. The present study was designed to exam- metal exposure (see 1-3, for reviews and references).ine the roles of MTs in cell proliferation using an in The physiological functions and regulation of the ex-vivo model of liver regeneration following partial hep-

pression of MT, which vary in different tissues andatectomy (PH) in rats. The levels of MT-I and MT-IIorgans (4), are still a matter of discussion. The controlwere studied with respect to regulation of prolifera-of MT synthesis occurs at the level of transcription,tive potential, cell cycle checkpoint activity, and oxi-which has been confirmed by measurement of mRNAdative stress in the rat PH model. We synthesized a 17-levels using cell-free translation systems and by directmer antisense phosphorothioate oligodeoxynucleotideassay using cDNA probes (5–7). MT genes are ex-(S-ODN), named aMT, complimentary to the start sitepressed in most tissues of most organisms, and they areof the MT-I mRNA sequence and an appropriate con-transcriptionally regulated by metals, glucocorticoids,trol. Both S-ODNs were administered intraperitone-and cytokines, indicating that MT synthesis is influ-ally at the dose of 5 mg/kg following 70% PH. MT be-

came induced 57.4 { 9.8-fold following PH and the said enced by stress stimuli (8, 9). In addition, transactingeffect became attenuated dramatically following ad- factors such as c-fos (10) and c-myc (11) have been alsoministration of aMT. In addition, PH rats treated with implicated as regulators of MT gene expression.aMT exhibited decreased rate of liver regeneration as Sequence-specific antisense oligonucleotides (anti-measured by expression of proliferating cell nuclear sense ODNs) have been used to delineate the regula-antigen and elevated cell cycle checkpoint activity as tion of MT synthesis. For example, Takeda et al. (12)determined by expression of p53. The results of these by using synthetic oligonucleotides with sequencesstudies suggest that MT isoforms with their high thiol complementary to the mRNA coding for human MT-IIcontents do play an important role in cellular func- have shown that a) inhibition of MT causes cells to dietions and especially during stressful states induced by from metal toxicity, b) MT possesses an essential gene,a broad range of mediators generating free radicals. and c) modification in oligonucleotide structures ex-q 1998 Academic Press hibit specificity in inhibiting MT synthesis. Moreover,Key Words: metallothionein; partial hepatectomy overexpression of MT causes resistance to certain anti-and liver regeneration; antisense oligonucleotides;

neoplastic agents (13).acute phase protein and oxidative stress.The role of MT in regulating cell cycle and modulat-

ing proliferating capacity has been studied in tumorcell lines (14–16). Furthermore, there occurs a translo-

Metallothionein (MT), an intracellular metallopro- cation of MT from the cytoplasm to the nucleus duringtein of low molecular weight (6,000-7,000), has a high S through G2-M phases of the cell cycle (17). Not onlycontent of cystenyl residues (33%), contains no aro- is zinc bound to MT but it also induces MT expressionmatic amino acids or disulfide bonds, and binds be- (2, 3). Zinc deficiency reduces the synthesis of MT (18)

and increases the generation of free radicals (19) whichin turn may suppress cellular progression through the1 Address correspondence and reprint requests to Manuchair cell cycle with an expected G1 to S phase block (13).Ebadi, Ph.D., Department of Pharmacology, University of Nebraska

Antisense ODNs, important tools in research andCollege of Medicine, Omaha, NE 68198-6260. Fax: 402-559-7495.E-mail: [email protected]. therapy (20), are short chains (10 to 50 bases) of de-

0006-291X/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 246, No. 3, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

oxynucleotides complimentary to a specific sequencein a segment of mRNA. Replacement of an oxygenwith a sulfur on each non bridging internucleosidephosphate produces a phosphorothioate (S-ODN),which is more resistant to nucleases and has only

FIG. 1. Sequences of MT-I and MT-II mRNAs from the ATG startminor differences in hydrogen bonding when com-site are alligned with that of the antisense oligonucleotide aMT. aMTpared to phosphodiesters (21, 22). Previous work inis completely complimentary to the MT-I sequence while it has twothis and other laboratories have demonstrated themismatches with the MT-II sequence (shown in bold typeface). The

ability of S-ODNs to reduce the expression of specific sequence of the control oligonucleotide OL-1 is also shown. Both aMTproteins in vivo in a sequence-specific manner (12, and OL-1 were synthesized with phosphorothioate chemistries (12,

23, 26).23, 24). Although the exact mechanisms of action arestill under investigation, decreased expression re-sults from inhibition of mRNA translation throughactivation of RNase H which cleaves RNA in a (Foster City, CA) with 1-mmol column supports. S-ODNs were pre-

pared by the cyanoethyl approach via phosphoramidite chemistry asRNA:DNA duplex (20). Pharmacokinetics of S-ODNselaborated by ABI user bulletin, no. 58, 1991, and described pre-in vivo have shown that these are not significantlyviously (29–31). The two S-ODN sequences used are exhibited indegraded and can be retrieved from animals as unde- Fig. 1. MT was designed to target MT-I mRNA at positions 1743–

graded material (25, 26). Furthermore, studies in 1759 (Genbank accession # M-11794). The control S-ODN OL-1 hadthis and other laboratories have shown the liver as no target sequence in the rat genome. Both S-ODNs were adminis-

tered at the dose of 0.2 mg/kg intraperitoneally immediately follow-the major site of accumulation for S-ODNs (26, 27).ing PH.The present study explores the role of MT in the

Induction of hepatic metallothionein isoforms I and II. The exper-process of liver regeneration, which is an in vivo modelimental rats were injected with 7.5 mg/kg of zinc sulfate, whereasof rapidly proliferating cells (28). Immunohistologicalthe control rats received an identical volume of physiological saline.studies have shown that MT isoforms are induced in The rats were decapitated 24 hr post-zinc treatment, and the livers

the regenerating liver following partial hepatectomy were removed and perfused with ice-cold nitrogen-saturated 0.01 M(PH), with the peak observed 24 hr following PH (17). Tris-HCl buffer, pH 7.5, containing 1 mM dithiothreitol. MT-I and

II were isolated and purified according to established procedures asThe use of S-ODNs targeted at MT can present adescribed by Ebadi (32) and Ebadi et al. (33).unique insight into the function of this metalloprotein

Measurement of metallothionein isoforms I and II (MT-I and MT-by causing a reduced induction of its expression in theII) using enzyme-linked immunosorbent assay (ELISA). MT iso-PH model. We have hypothesized that inhibition of ele-forms I and II were measured in liver homogenates using 96 wellvated levels of MT during liver regeneration will result ELISA plates essentially as described by Garvey (34). Anti-rabbit

in an increase in reactive oxygen intermediates (ROI) polyclonal antibodies which cross react with rat MT-I and MT-II (33)levels in the regenerating liver and thereby facilitate were purchased from Alpha Gamma Labs (Sierra Madre, CA). The

secondary antibody was goat anti-rabbit IgG with horseradish perox-cell cycle arrest and diminished liver regeneration.idase label (Sigma). The color generated by horseradish peroxidaseThrough the use of S-ODNs targeted at MT-I in thesubstrate was measured on a microplate reader.regenerating liver, we have shown that inhibition of

Measurement of hepatic metallothionein mRNA by in situ hybrid-the induction of MT decreases liver regeneration andization. By using MT-I cDNA as a probe and by applying Northerncauses higher oxidative stress.blot analyses, we measured hepatic MT-I mRNA in the zinc-treatedand, as a control, saline-treated rats (35).

MATERIALS AND METHODS Measurement of thiobarbituric acid reactive substances (TBARS).Lipid peroxidation is used as a direct indicator of oxidative stress.Lipid peroxidation brought about by an inhibition of synthesis of MTAnimals. Male Sprague Dawley rats (200-225 gm) were used for

the study and obtained from Harlan Sprague Dawley, Indianapolis, isoforms was measured in the liver homogenates by a modificationof the method of Esterbauer and Cheeseman (36). After precipitatingIN. Animals were housed in clear plastic cages in the AAALAC-

approved animal facility at the University of Nebraska Medical Cen- the proteins in the liver homogenates with an equal volume of 10%trichloroacetic acid, the samples were centrifuged at 1000 rpm forter under a controlled temperature and humidity climate and 12 hr

light/dark cycles. Rats had access to tap water and standard rat chow 10 minutes. One milliliter of the supernatant was then incubatedwith one milliliter of 0.67% thiobarbituric acid (TBA, Sigma) at 1007C(Harlan Teklad, Madison, WI) ad libitum.for 10 minutes. Lipid peroxides do react with TBA (collectivelyPerforming partial hepatectomy (PH). Rats were anesthetized termed TBARS) to produce a colored TBA-MDA product, which waswith methoxyflurane (Mallinckrodt, Mundelein, IL) and subjected to measured at an absorbency maximum of 532 nm. The background70% partial hepatectomy according to a technique described by Hig- was corrected by subtracting the average absorbency at 508 nm andgins and Anderson (28). An incision was made on the abdomen along 556 nm from the absorbency at 532 nm (37). A standard curve wasthe median line under aseptic conditions followed by ligature and generated using various concentrations of MDA (Sigma) and 0.67%excision of the medial and left lateral lobes of the liver. Rats that TBA using a similar protocol. It should be noted that there has beendid not receive PH underwent a sham surgery involving gentle ma- some concern in the literature that the assay conditions for TBARSnipulation of the medial and left lateral lobes of the liver. determination may result in additional decomposition of lipids in thesample, resulting in artificially high values (37, 38). In order to ac-Synthesis, purification, and administration of phosphorothioate

oligodeoxynucleotides (S-ODN). All chain-extension syntheses were count for this possibility, all experimental results were adjusted us-ing negative controls.performed on an Applied Biosystems Model 380B DNA synthesizer

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Immunoblot analyses of p53, PCNA, and NADPH reductase. Pro-tein obtained from fifty micrograms of total liver homogenate wereseparated in a 10% SDS-PAGE gel. Rainbow protein molecularweight markers (Amersham Corp., Arlington Heights, IL) were usedas markers, which were transferred to Immobilon-P transfer mem-branes (Millipore Corp., Bedford, MA). After blotting for one hr with1% BSA, membrane blots were probed with respective primary anti-bodies, namely p53, PCNA (both from Oncogene Sciences, La Jolla,CA) and NADPH Reductase antibodies (Gentest, Woburn, MA). Afterwashing, the blots were probed again using secondary antibodieswith horseradish peroxidase label (Pierce, Rockford, IL). ECL re-agents (Amersham Corp.) were used to produce chemiluminescenceand the blots were exposed on Kodak x-ray film. Band intensitieswere determined by a Molecular Dynamics Personal Densitometer(Sunny Vale, CA) using ImageQuant v3.3 software (Molecular Dy-namics).

Microsomal isolation. Hepatic microsomes were prepared by amodification of the method of Franklin and Estabrook (39). Liverswere perfused immediately with 10 ml ice cold saline via the portal FIG. 2. Zinc sulfate induces the synthesis of hepatic metallothio-vein, minced, homogenized in 10 ml 0.25 M sucrose (Sigma), and nein isoforms I and II which bind 0.40 and 0.45 mg zinc/ml elution,centrifuged at 8000 rcf for 20 min at 47C in a Sorvall RC2-B centri- respectively.fuge (Dupont, Wilmington, DE). The supernatants were centri-fuged at 100,000 rcf for 45 min at 47C in a Sorvall OTD55B ultra-centrifuge (Dupont). The pellets were resuspended in 20 ml 0.15MKCl (Sigma) and centrifuged at 100,000 rcf for 45 min at 47C. The bencies were compared to a standard curve generated from knownfinal pellets were resuspended in an equivalent volume of buffer concentrations of formaldehyde. Activities were recorded as mmol(10 mM Tris-acetate, 1 mM EDTA, 20% glycerol; Sigma; pH Å 7.4) formaldehyde/mg protein/min.and stored at 0807C.

Statistical analysis. With the exception densitometric analysesMeasurement of cytochrome P4502E1. The activity of CYP2E1 in all data were reported as mean { standard error of the mean (S.E.)

the microsomes was measured using p-nitrophenol hydroxylase as determined by the computer program Instat 2 (GraphPad, San(PNP) assay (40, 41). An aliquot of 1 mg microsomal protein was Diego, CA). The P values were also calculated with the same softwaremixed with 0.01 ml 20 mM p-nitrophenol (Sigma), and 0.05 ml 60 using Tukey multiple comparison test. Standard curves and graphsmM NADPH (Sigma) and diluted to 1 ml with 0.1 M potassium were generated using Prism v2 (GraphPad).phosphate buffer (pH Å 7.4). The samples were incubated for 10 minat 377C, mixed with 0.25 ml 15% trichloroacetic acid (Sigma), and

RESULTSwere centrifuged at 15000 rcf for 4 min in a Hermle Z 230 M centri-fuge (National Labnet Co., Woodsbridge, NJ). A 1 ml sample wasadded to a new set of tubes containing 0.1 ml 10 N NaOH and mixed Administration of zinc sulphate induced the synthe-for 10 sec. Each sample was then read at 515 nm on a Ultraspec III

sis of MT-I mRNA (Figs. 2 and 3) and enhanced the(Pharmacia). Enzyme activity was recorded as OD/mg protein/min.synthesis of MT-I mRNA (Fig. 4) and these findings are

Measurements of the activities of cytochromes P4501A1 and 2B2 characteristic of hepatic MT isoforms as documented(CYP1A1 and 2B2). The activities of microsomal CYP1A1 and 2B2previously (see refs. 1, 2). Partial hepatectomy in-were determined by using ethoxyresorufin-o-dealkylation assaycreased the levels of MT-I and II and the said effects(EROD) or pentoxyresorufin-o-dealkylation assay (PROD), respec-

tively (42). An aliquot of 1 mg microsomal protein in 1 ml, 0.1 M were attenuated by administration of antisense oligo-potassium phosphate buffer (pH Å 7.4), 1 ml 2 mM 5-ethoxy- or 5- nucleotide but not control oligonucleotide (Fig. 5).pentoxyresorufin (Pierce, Rockford, IL), and 0.02 ml 60 mM NADPHwere mixed and incubated for 10 min at 377C. The mixture was then Expression of PCNA. Proliferating cell nuclear an-added to a 2 ml cuvette and read on a RF5000U spectrofluoropho- tigen (PCNA) is a component of the DNA polymerasetometer (Shimadzu, Columbia, MD) using an excitation wavelength machinery and its levels in a tissue are an accurateof 530 nm and emission wavelength of 585 nm. Concentrations of

reflection cellular proliferation (58). PCNA levels wereunknowns were calculated from a standard curve of resorufin (Pierce,measured in liver homogenates by immunoblotting andRockford, IL) standards. Results were recorded in pmol resorufin/

mg protein/min. a representative blot is shown along with quantitativedensitometric analysis (Fig. 6). PCNA levels wereMeasurement of cytochrome P4503A2 (CYP3A2). By using eryth-

romycin demethylation assay (ERDEM) the activity of CYP3A2 was found be similar in both saline and OL-1 treated shammeasured (43). The Samples were prepared by mixing 1.0 mg of PH rats, while aMT treated sham PH rats showed amicrosomes, 0.4 mM erythromycin, and 1.0 mM NADPH in a final somewhat lesser band density. There occurred a 2.6-volume of 1 ml in 0.1 M potassium phosphate buffer (pH Å 7.4). The

fold increase in PCNA detection in saline treated PHsamples were incubated for 15 min at 377C. The formaldehyde wasrat livers, an expected result caused by regenerationthen assayed by the colorimetric method developed by Nash (44).

The samples were mixed with 0.5 ml 17% perchloric acid (Sigma) of liver following PH. There occurred a similar increaseand centrifuged on the Hermle microcentrifuge at 15000 rcf for 5 in OL-1 treated 24h PH rat livers. However, livers frommin. The samples were placed in a new tube and mixed with 0.4 ml 24h PH aMT treated rats completely failed to induceNash reagent (0.02 M 2,4 pentanedione, 0.6% v/v glacial acetic acid,

PCNA levels, suggesting a loss of regenerative capacityand 3.9 M ammonium acetate) and incubated at 707C for 20 min.The samples were read on the spectrophotometer at 412 nm. Absor- in the livers of these rats. The level of an unrelated

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TBARS in aMT treated rats suggests a certain degreeof oxidative stress. A more dramatic difference inTBARS levels were seen in 24 hr PH rats. Firstly, thereoccurred a 4.24-fold increase in TBARS between salinetreated sham PH and 24 hr PH rats, suggesting thatincreased oxidative stress was associated with liver re-generation. More convincingly, there occurred a 19.48-fold increase in TBARS with aMT treatment in 24 hrPH rats, indicating that MT levels are related to thedegree of oxidative stress in liver regeneration.

Decreased MT induction causes slower functional re-covery of the regenerating liver. The functional capac-ity of regenerating liver was assessed in microsomalpreparations of the remnant regenerating livers. Cyto-chromes P450 (CYP) 1A1/2 and 2B1/2 enzyme activitieswere determined by ethoxy and pentoxyresorufin O-dealkylase activities (EROD/PROD), respectively. Ac-tivities were recorded in picomoles of resorufin per mgof total protein per minute. CYP 2E1 was determinedby p-nitrophenol (PNP) activity. Activity was recordedas optical density (O.D.) per mg of total protein perminute. Erythromycin demethylase (ERDEM) wasused to measure CYP 3A2 activity which was expressedas mmoles of formaldehyde per mg of total protein perminute. These data are presented in Fig. 9 as percent

FIG. 3. A typical profile of the zinc-induced hepatic metallothio-of controls. The activities expressed as 100% are asnein I and II exhibiting two peaks on HPLC with retention times offollows (n Å 10 for all):15.69 (MT-I, top panel) and 16.47 (MT-II, bottom panel) minutes,

respectively.EROD: 34.8 { 3.2 picoM of resorufin/mg protein/

minutePROD: 6.4 { 0.4 picoM of resorufin/mg protein/protein, NADPH reductase, was found to be similar in

minuteliver homogenates of all treatment groups.ERDEM: 107 { 11 mmoles of formaldehyde/mg pro-

Expression of p53. Levels of the G1-S cell cycle tein/minutecheckpoint protein p53 were measured in liver homoge- PNP: 0.9 { 0.1 O.D. units/mg protein/minutenates to further characterize the role of MT in cell pro- (Fig. 9).liferation (Fig. 7). p53 was undetectable in immu-noblots of liver homogenates of sham PH rats, irrespec- These data indicate that the levels for all of the CYPtive of the treatment regimens. As expected, there enzymes studied declined by more than half at 24 hroccurred an induction in p53 expression in livers of all post PH. There were no statistically significant differ-groups of 24h PH rats corresponding with increased ences in CYP recovery between OL-1 and saline treatedproliferation associated with liver regeneration. This rats for any of the enzyme studied. aMT treatmentinduction in p53 expression was similar in both saline in every case (except CYP 2E1) caused a significantand OL-1 treated 24 hr PH rats. However, the induc- decrease in functional recovery of CYP enzymes 24 hrtion was further increased from 712 to 1012 densito- post PH.metric units in 24 hr PH rats treated with aMT, sug-gesting an elevated cell cycle checkpoint activity in this DISCUSSIONgroup. This finding correlates well with the decreasedproliferation associated with aMT treatment and is ap- The present study has underlined the importance ofparent from the PCNA data (Fig. 7). MT isoforms I and II in the process of cell proliferation

in vivo. We chose to investigate the role of MT isoformsOxidative stress measurements. Lipid peroxidationwas measured in liver homogenates as an index of oxi- 24 hr following PH based on a preliminary report that

MT is induced following 70% PH in rats (17, 45). Ourdative stress using the thiobarbituric acid reactive sub-stances (TBARS) assay (Fig. 8). Saline treated sham results are in general agreement with these studies as

we found about 57-fold elevation in MT-I and about 11-PH rats had TBARS level of 3.7{ 2.0 pM/mg. Althoughthere was no statistical difference between any of the fold elevation in MT-II levels in liver homogenates 24

hr following 70% PH in rats (Figure 5). MT isoformssham PH treatment groups, a slightly higher value for

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FIG. 4. A typical pseudocolor image of rat liver demonstrating the regional localization of zinc-induced metallothionein I mRNA asdetermined by in situ hybridization histochemistry and autoradiography (35).

are found in low concentrations in most tissues but are but a role in cell proliferation has been proposed (51,54). A relationship between S-phase of cell cycle andinduced following exposure to a variety of compounds

like zinc or other heavy metals (46, 47), glucocorticoids MT elevation (55, 56) has further made a case for arole of metallothionein in proliferating cells.(48) and endotoxin (49). High levels of MT expression

are normally observed in tissues of newborn (50) andfetal (51) rat as well as neoplastic tissue (52, 53). Thebiological significance of MT induction in absence ofchallenge by non metallic compounds remains unclear,

FIG. 5. The effects of Sham operation and saline treatment (NPHsal), Sham operation and control oligonucleotide treatment (NPHOL-1), Sham operation and antisense oligonucleotide treatment(NPH aMT), and partial hepatectomy following treatments with con- FIG. 6. A representative SDS-PAGE immunoblot of proliferating

cell nuclear antigen (upper panel) and NADPH Reductase (lowertrol and antisense oligonucleotide are shown in this figure. Partialhepatectomy enhanced the levels of MT-I and II and the said induc- panel) in liver homogenates. The various treatment modalities are

indicated. Results of the quantitative densitometric analysis of thetion was attenuated dramatically with administration of antisenseoligonucleotide. The effects of 24 hr a MT is significant at p õ 0.005 bands from the immunoblots are also tabulated. The densitomemtric

data are in arbitrary units.level.

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cycle checkpoint protein that is related to the progressof cells from the G1 to S phase of the cell cycle (Fig. 6).Our studies with various parameters of cell prolifera-tion indicate that an induction of MT plays a permis-sive role in the passage of cells through the cell cycleand an interference with this induction can lead to re-duced regenerative potential.

The rapidly proliferating cells of the regeneratingliver are extremely susceptible to oxidant damage. Re-cent studies have shown a role for MT as a scavengerof free radicals induced by oxidative stress (59–61).Furthermore, in vivo studies have demonstrated thatzinc-treated mice exhibit an increased resistance to X-irradiation (62). Since much of the DNA damage causedby ionizing radiation is thought to result from genera-tion of hydroxyl radicals, MT is now considered to bean potent endogenous scavenger of hydroxyl radicals.Electromagnetic spin resonance studies suggest thatin addition to hydroxyl, MT is also involved in scaveng-ing of superoxide and organic radicals (63). We mea-

FIG. 7. A representative SDS-PAGE immunoblot of p53 (upper sured TBARS in homogenates of regenerating livers topanel) and NADPH Reductase (lower panel) in liver homogenates. examine the extent of lipid peroxidation and the effectThe various treatment modalities are indicated. Results of the quan-

of aMT treatment on the same (Fig. 8). We found thattitative densitometric analysis of the bands from the immunoblotsthe process of regeneration itself was associated withare also tabulated. The densitomemtric data are in arbitrary units.an elevated oxidative stress, but inhibition of MT in-duction during regeneration caused a further increasein levels of TBARS. This observation is in keeping withIt is thought that the most important physiological

role of MT is as a zinc binding protein (57). While any the radical scavenger role of MT. Thus, an elevation ofMT during the process of regeneration, protect againstessential metal must be indispensable for growth and

differentiation, zinc in particular has a multiplicity of oxidative reaction.Along with greater degrees of oxidant stress, the PHbiological functions. It stabilizes the structures of vari-

ous proteins, DNA, RNA, ribosomes and membranes, rats treated with aMT also showed less degree of liverregeneration than seen in OL-1 treated control rats, asin addition to being a component of several DNA and

RNA polymerases (56). The trauma of surgery itself evident from the PCNA expression data. We postulatethat this is a result of increased DNA damage causedcould also affect MT levels. Although we did not observe

any significant elevation in either MT-I or MT-II in by oxidant stress. The two lines of evidence that sub-stantiate this contention is that these rats have muchlivers from sham operated rats 24 hr after the surgery

(data not shown), it has been previously reported thatMT levels are elevated in serum and depressed in liver18 to 48 hr following adrenalectomy (57). The lack ofdifference in MT I and II values between saline-treatedsham PH and normal rats suggests that the MT induc-tion 24 hr post PH is associated with loss of liver and/or regeneration rather than the stress of surgery itself.

We were able to significantly modulate MT elevationfollowing PH and reduce the induction of the majorisoform, MT-I, from about 57-fold to a mere 10-fold bythe use of antisense S-ODN aMT (Figs. 1 and 5). Thedosage and route of administration of the S-ODNs werebased on a previous study from our laboratory (23). Inthe present study, we have seen that the preventionof MT induction with aMT during liver regenerationcauses a concomitant decrease in the induction of

FIG. 8. Oxidative stress was measured by determination of Thio-PCNA, which is a component of the DNA polymerasebarbituric Acid Reactive Substances (TBARS) in the liver homoge-

d machinery and an accurate indicator of the degree of nates. The results are expressed in pM/mg of total homogenate pro-proliferation (Fig. 6 and ref. 58). The use of aMT also tein. * indicates a statistical difference of põ 0.05 compared to saline

treated animals within the respective PH or NPH group.correlated with increased expression of p53, the cell

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FIG. 9. Liver CYP activities were assessed in the microsome fractions. CYPs 1A1 and 2B1 enzyme activities were determined by ethoxyand pentoxyresorufin O-dealkylase activities (EROD/PROD), respectively. Activities were recorded in picomoles of resorufin per mg of totalprotein per minute (panels A & B). Erythromycin demethylase (ERDEM) was the measure of CYP 3A2 activity and was recorded as mmoleof formaldehyde per mg of total protein per minute (panel C). CYP 2E1 was determined by p-nitrophenol (PNP) activity (panel D). Activitywas recorded as optical density (OD) per mg of total protein per minute. The data are presented in various panels as percent of control.

higher levels of p53 in their liver homogenates (Fig. 7). ACKNOWLEDGMENTSp53 is known to be induced following DNA damage and

The authors express their appreciation to Mrs. Lori Ann Clapperprevents the progression of cells through the cell cycle.for her excellent secretarial skills. This study was completed in partThrough its actions mediated by p21Waf1, p53 arrestsby grants from USPHS numbers NS34566 and GM54871. V. Arora

cells in the G1-S phase till the damaged DNA is re- received fellowship support from Nebraska Research Initiative inpaired (64). The second line of evidence is that there is Biotechnology.a translocation of MT from the cytosol to the nucleuswithin hours following PH with the peak observed at REFERENCES24 hr (65). MT may donate a hydrogen atom to a radical

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