Silymarin and silibinin cause G1 and G2-M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin.

Here, we assessed and compared the anticancer efficacy and associated mechanisms of silymarin and silibinin in human prostate cancer (PCA) PC3 cells; silymarin is comprised of silibinin and its other stereoisomers, including isosilybin A, isosilybin B, silydianin, silychristin and isosilychristin. Silymarin and silibinin (50-100 microg/ml) inhibited cell proliferation, induced cell death, and caused G1 and G2-M cell cycle arrest in a dose/time-dependent manner. Molecular studies showed that G1 arrest was associated with a decrease in cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)4, CDK6 and CDK2 protein levels, and CDK2 and CDK4 kinase activity, together with an increase in CDK inhibitors (CDKIs) Kip1/p27 and Cip1/p21. Further, both agents caused cytoplasmic sequestration of cyclin D1 and CDK2, contributing to G1 arrest. The G2-M arrest by silibinin and silymarin was associated with decreased levels of cyclin B1, cyclin A, pCdc2 (Tyr15), Cdc2, and an inhibition of Cdc2 kinase activity. Both agents also decreased the levels of Cdc25B and cell division cycle 25C (Cdc25C) phosphatases with an increased phosphorylation of Cdc25C at Ser216 and its translocation from nucleus to the cytoplasm, which was accompanied by an increased binding with 14-3-3beta. Both agents also increased checkpoint kinase (Chk)2 phosphorylation at Thr68 and Ser19 sites, which is known to phosphorylate Cdc25C at Ser216 site. Chk2-specific small interfering RNA largely attenuated the silymarin and silibinin-induced G2-M arrest. An increase in the phosphorylation of histone 2AX and ataxia telangiectasia mutated was also observed. These findings indicate that silymarin and silibinin modulate G1 phase cyclins-CDKs-CDKIs for G1 arrest, and the Chk2-Cdc25C-Cdc2/cyclin B1 pathway for G2-M arrest, together with an altered subcellular localization of critical cell cycle regulators. Overall, we observed comparable effects for both silymarin and silibinin at equal concentrations by weight, suggesting that silibinin could be a major cell cycle-inhibitory component in silymarin. However, other silibinin stereoisomers present in silymarin also contribute to its efficacy, and could be of interest for future investigation.

Structural determinants outside of the leucine zipper influence the interactions of CREB and ATF-2: interaction of CREB with ATF-2 blocks E1a-ATF-2 complex formation.

Dimerization of leucine zipper-containing proteins has been associated characteristically with the formation of a coiled-coil structure between two compatible leucine zipper motifs. In the present study we demonstrate the association of the leucine zipper of cAMP response element-binding protein (CREB) with a zinc finger motif of ATF-2. The association of the CREB leucine zipper with the ATF-2 zinc finger is stabilized if the ATF-2 leucine zipper is intact, implying that the preferred interactive structure of ATF-2 juxtaposes the amino-terminal zinc finger motif of this protein with the carboxy-terminal leucine zipper of this same protein. Furthermore, we demonstrate that the association of the CREB leucine zipper with the ATF-2 zinc finger in vitro blocks the association of the adenoviral E1a protein with ATF-2. Similarly, overexpression of full-length CREB, or a truncated version of this protein corresponding to the carboxy-terminal 74 amino acids that make up the DNA-binding and dimerization domains, can block the ATF-2-mediated transcriptional stimulation by E1a in vivo. Mutation of the ATF-2 zinc finger motif stimulates DNA binding of this protein, and abolishes interactions with E1a and CREB proteins. These results demonstrate that the structural conformation of ATF-2 is critical for DNA binding and protein-protein interactions and, further, that leucine zippers can mediate protein-protein interactions with structural motifs other than leucine zippers.

Modification of DNA topoisomerase II activity via direct interactions with the cyclic adenosine-3',5'-monophosphate response element-binding protein and related transcription factors.

DNA topoisomerase II (topo II) is an essential nuclear enzyme which catalyzes the interconversions of various forms of DNA. As predicted from the human topo II cDNA, the enzyme contains a potential leucine zipper protein dimerization motif. We therefore tested whether topo II could enter protein-protein interactions with other better characterized leucine zipper-containing proteins and determined if these interactions could modify topo II enzymatic activity in vitro. By far Western analyses, a large C-terminal fragment of human topo II was shown to interact with the DNA binding and dimerization regions of either cAMP response element binding protein (CREB) or the activating transcription factor-2. The C-terminal topo II fragment also interacted with full-length c-Jun, but not with full-length c-Fos. Using CREB as a prototype, the effect of this interaction on various topo II catalytic activities was assessed in vitro. CREB, at a 1- to 10-fold molar excess relative to topo II, inhibited site-specific DNA cleavage activity on a 242-base pair fragment of the human alpha-glycoprotein hormone subunit gene promoter. Very high CREB concentrations (400-fold excess) apparently inhibited topo II DNA relaxation activity, but this result was likely a direct effect of CREB on the topology of the DNA substrate. More interestingly, a 10-fold molar excess of CREB stimulated topo II decatenation activity, the essential function of this enzyme in cell division. This stimulatory effect could also be elicited by c-Jun, which interacts with topo II, but not by c-Fos, which does not bind topo II in our in vitro assay. Since similar amounts of CREB reduced the abundance of topo II DNA cleavage products from the human alpha-CG promoter yet also stimulated decatenation activity, it can be concluded that either: 1) CREB stimulated the religation rate of topo II; or 2) CREB directed topo II to a new cleavage site present on the decatenation substrate but not present on the limited alpha-CG promoter. The structural requirements for topo II protein-protein interactions were also investigated. Site-directed mutations which destroyed the putative topo II leucine zipper did not disrupt topo II protein-protein interactions. Since the putative leucine zipper in topo II does not appear to mediate protein-protein interactions, we propose that an alternate as yet uncharacterized structure is involved in the association of topo II with itself and other regulatory proteins.

Activating transcription factor-2 DNA-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases.

Recent studies have detailed the ability of activating transcription factor-2 (ATF-2) to mediate adenoviral E1a stimulation of gene expression; however, an endogenous regulator for the transcriptional activity of this protein has not been described. To characterize the regulation of ATF-2 activity, we have expressed full-length and truncated peptides corresponding to various regions of the ATF-2 protein in bacteria and the baculovirus insect cell system. Bacterially expressed truncated (350-505) but not full-length ATF-2, was able to bind a consensus cAMP response element-containing oligonucleotide, suggesting the N-terminal moiety may serve as a negative regulator of DNA-binding activity. In contrast, the full-length ATF-2 protein expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus was fully competent to bind DNA. Protein phosphatase 2A reversed the DNA-binding activity by dephosphorylating the ATF-2 polypeptide. Microtubule-associated protein kinase catalyzed the phosphorylation and stimulated the DNA-binding activity of bacterially expressed full-length ATF-2. Phosphopeptide mapping of phosphorylated ATF-2 proteins identified a single peptide in the N-terminal moiety of ATF-2 phosphorylated by p42 or p54 microtubule-associated protein kinase. Therefore, we propose that phosphorylation of this regulatory site is sufficient to induce an allosteric structural change in the ATF-2 protein, which allows dimerization and subsequent DNA binding.

Herbs as medicines.

Herbs and related products are commonly used by patients who also seek conventional health care. All physicians, regardless of specialty or interest, care for patients who use products that are neither prescribed nor recommended. Some herbs have been extensively studied, but little is known about others. When a patient asks for advice regarding the use of a particular herb, how should a physician respond? Similarly, how does a physician determine if a patient's symptoms are caused by a "remedy"? This review attempts to answer these questions by investigating pertinent definitions, the history of herbs in medicine, epidemiology and prevalence of herbal use, and relevant psychosocial issues.

The histone deacetylase inhibitor sodium butyrate induces DNA topoisomerase II alpha expression and confers hypersensitivity to etoposide in human leukemic cell lines.

The differentiating agent and histone deacetylase inhibitor, sodium butyrate (NaB), was shown previously to cause a transient, 3-17-fold induction of human DNA topoisomerase II alpha (topo II alpha) gene promoter activity and a 2-fold increase in topo II alpha protein early in monocytic differentiation of HL-60 cells. This observation has now been extended to other short chain fatty acids and aromatic butyrate analogues, and evidence is presented that human topo II alpha promoter induction correlates closely with histone H4 acetylation status. Because increased topo II alpha expression is associated with enhanced efficacy of topo II-poisoning antitumor drugs such as etoposide, the hypothesis tested in this report was whether NaB pretreatment could sensitize HL-60 myeloid leukemia and K562 erythroleukemia cells to etoposide-triggered DNA damage and cell death. A 24-72 h NaB treatment (0.4-0.5 mM) induced topo II alpha 2-2.5-fold in both HL-60 and K562 cells and caused a dose-dependent enhancement of etoposidestimulated, protein-linked DNA complexes in both cell lines. At concentrations with minimal effects on cell cycle kinetics (0.4 mM in HL-60; 0.5 mM in K562), NaB pretreatment also modestly enhanced etoposidetriggered apoptosis in HL-60 cells, as determined morphologically after acridine orange/ethidium bromide staining, and substantially increased K562 growth inhibition and poly(ADP-ribose)polymerase cleavage after etoposide exposure. Therefore, a temporal window may exist whereby a differentiating agent may sensitize experimental leukemias to a cytotoxic antitumor agent. These results indicate that histone deacetylase inhibitors should be investigated for etoposide sensitization of other butyrate-responsive hematopoietic and nonhematopoietic tumor lines in vitro and in vivo.

Rhythm annotation and interobserver reproducibility of measures of heart rate variability.

Interobserver reproducibility is high for time domain and power spectral measures of heart rate variability, with greater reproducibility for low-frequency measures, and especially for the standard deviation of the 5-minute RR intervals over 24 hours, than for high-frequency measures. Overall interobserver variability of < 8% for these measures is largely (50% to 75%) explained by interobserver differences in annotation of supraventricular ectopy and sinus arrhythmia.

Catalytic inhibition of human DNA topoisomerase IIalpha by hypericin, a naphthodianthrone from St. John's wort (Hypericum perforatum).

St. John's wort (Hypericum perforatum) is the most widely used herbal medicine for the treatment of depression. However, concerns have arisen about the potential of its interaction with other drugs due to the induction of cytochrome P450 isozymes 1A2 and 3A4 by the components hypericin and hyperforin, respectively. Structurally similar natural products are often employed as antitumor agents due to their action as inhibitors of DNA topoisomerases, nuclear enzymes that modify DNA during cellular proliferation. Preliminary findings that hypericin inhibited the DNA relaxation activity of topoisomerase IIalpha (topo II; EC 5.99.1.3) led us to investigate the mechanism of enzyme inhibition. Rather than stabilizing the enzyme in covalent complexes with DNA (cleavage complexes), hypericin inhibited the enzyme prior to DNA cleavage. In vitro assays indicate that hypericin is a potent antagonist of cleavage complex stabilization by the chemotherapeutics etoposide and amsacrine. This antagonism appears to be due to the ability of hypericin to intercalate or distort DNA structure, thereby precluding topo II binding and/or DNA cleavage. Supporting its non-DNA damaging, catalytic inhibition of topo II, hypericin was shown to be equitoxic to both wild-type and amsacrine-resistant HL-60 leukemia cell lines. Moreover, hypericin was incapable of stimulating DNA damage-responsive gene promoters that are activated by etoposide. As with the in vitro topo II assay, antagonism of DNA damage stimulated by 30 microM etoposide was evident in leukemia cells pretreated with 5 microM hypericin. Since many cancer patients experience clinical depression and concomitantly self-medicate with herbal remedies, extracts of St. John's wort should be investigated further for their potential to antagonize topo II-directed chemotherapy regimens.

NF-M trans-activates the human DNA topoisomerase II alpha promoter independently of c-Myb in HL-60 cells.

Identifying transcriptional regulators of DNA topoisomerase II alpha (topo II alpha) is essential to decipher the mechanisms underlying leukemia cell resistance to topo II-directed antitumor drugs. We have previously reported that the proto-oncogene transcription factor c-Myb transactivates the topo II alpha promoter in several hematopoietic cell lines. Currently, we investigate whether NF-M, a C/EBP beta family member, cooperates with c-Myb in activating topo II alpha transcription. Although NF-M is the most efficacious trans-activator of topo II alpha that we have examined (approximately 38-fold over basal), NF-M does not appear to be involved in the endogenous transcriptional regulation of topo II alpha. Interestingly, we report that the sodium butyrate-dependent induction of the topo II alpha promoter observed previously appears to be mediated by c-Myb, independent of NF-M.

Drug sensitivity of heat-resistant mouse B16 melanoma variants.

Induction of transient thermotolerance by heat or other cytotoxic stressors has been reported to confer a moderate degree of drug resistance to tumor cells in vitro. In this study, a genetically stable, heat-resistant mouse B16 melanoma variant (W-H75) was tested for its sensitivity to various cytotoxic and antiproliferative agents. The heat-resistant W-H75 cells displayed a moderate two- to threefold resistance to doxorubicin, VP-16, VM-26, colchicine, cis-dichlorodiammineplatinum(II), HgCl2, and CdCl2. Marginal resistance to 4'(9-acridinylamino)methanesulfon-m-anisidide vinblastine, 1,3-bis(2-chloroethyl)-1-nitro-sourea, and NaAsO2 was observed, while no difference in sensitivity to the anticancer drugs, actinomycin D and camptothecin, was observed. Although W-H75 cells were generally more resistant than the parental cells to most of the agents that were tested, they were collaterally sensitive to the antimetabolites methotrexate and 6-mercaptopurine. Resistance of the W-H75 cells to epipodophyllotoxins and anthracyclines was not due to differences in steady-state drug accumulation. For the epipodophyllotoxin VP-16, resistance may be related to a relative decrease in the number of drug-induced DNA strand breaks in W-H75 cells. However, no difference in DNA strand breakage was observed between W-H75 and parental cells which were treated with doxorubicin, suggesting that resistance to this drug occurred by a different mechanism. The possible involvement of glutathione and glutathione S-transferase in resistance was also investigated. The glutathione content in W-H75 cells was 35% higher than that in the parental line. However, glutathione S-transferase activity appeared to be identical in both cell lines. Two other heat-resistant B16 melanoma variants, B-H103 and R-H92, were also tested for sensitivity to doxorubicin and VP-16. In contrast to the W-H75 cells, these two heat-resistant variants were hypersensitive to doxorubicin. The B-H103 cells were also hypersensitive to VP-16. This study suggests that selection for cellular resistance to heat may result in cells that have an altered sensitivity to drugs.

A multifunctional prokaryotic protein expression system: overproduction, affinity purification, and selective detection.

A series of plasmid vectors, pRSET A, B, and C, have been developed for high-level protein expression in prokaryotes and have been characterized. Based upon the T7 RNA polymerase-driven pET system, the pRSET vectors encode recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni(2+)-affinity resins, a tyrosine residue for radioiodination, and an enterokinase proteolytic cleavage site for leader peptide removal. Monoclonal antibodies (MAbs) to two epitopes on the leader peptide, which also contains amino acids 1-12 of the T7 gene 10 major capsid protein, were developed and provide for universal immunological detection of pRSET-expressed fusion proteins. Subcloning of protein-encoding DNA is facilitated by an 11-site polylinker which is offset for all three ribosomal reading frames, and an f1(+) origin of DNA replication permits single-stranded DNA synthesis for site-directed mutagenesis protocols. Representative fusion proteins overexpressed in Escherichia coli were successfully purified under both denaturing and nondenaturing conditions by single-step Ni2+ affinity chromatography. Purification was independent of recombinant protein solubility in sonicated or freeze-thawed E. coli lysates. Isolation of MAbs for selective recognition of either of two leader peptide epitopes was demonstrated by immunoprecipitation, but this selectivity was less evident under conditions for Western blotting. In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.

Stability of lipid/DNA complexes during agitation and freeze-thawing.

It is well established that cationic liposomes facilitate the delivery of DNA and offer substantial advantages over viral-based delivery systems. However, these synthetic vectors readily aggregate in liquid formulations which in clinical trials requires preparation of lipid/DNA complexes at the bedside immediately before injection. This temporal requirement could be eliminated if complexes were formulated as stable preparations that could be shipped, stored, and administered as needed. To this end, our study investigates the stability of lipid/DNA complexes during physical stresses that might be encountered during shipping and storage, i.e., agitation and freeze-thawing. Our data show that agitation significantly reduces transfection rates in complexes prepared with three different commercially available lipid formulations. Additional experiments indicate that slow freezing is more damaging than rapid freezing, and that sucrose is able to preserve transfection and complex size during freeze-thawing. These results are consistent with previous reports and demonstrate that frozen formulations may be suitable for maintaining transfection rates of lipid/DNA complexes. Under certain conditions, we observe a reproducible 3-fold increase in transfection after freeze-thawing that is prevented by high concentrations of sucrose. Together, these data suggest that physical stresses can alter structural characteristics of lipid/DNA complexes that can markedly affect rates of DNA delivery.

Preparation and in vitro characterization of gentamycin-impregnated biodegradable beads suitable for treatment of osteomyelitis.

A new method for preparing poly(L-lactide) (PLA) biodegradable beads impregnated with an ionic aminoglycoside, gentamycin, is described. The process employs hydrophobic ion pairing to solubilize gentamycin in a solvent compatible with PLA, followed by precipitation with a compressed antisolvent (supercritical carbon dioxide). The resulting precipitate is a homogeneous dispersion of the ion-paired drug in PLA microspheres. The microspheres are approximately 1 microm in diameter and can be compressed into beads (3-6 mm in diameter) strung on surgical sutures for implantation. The bead strings exhibit no significant change in release kinetics upon sterilization with a hydrogen peroxide plasma (Ster-Rad). The kinetics of gentamycin release from the PLA beads are consistent with a matrix-controlled diffusion mechanism. While nonbiodegradable poly(methyl methacrylate) (PMMA) beads initially release gentamycin in a similar manner, the drug release from PMMA ceases after 8 or 9 weeks, while the PLA beads continue to release drug for over 4 months. Moreover, only 10% of the gentamycin is released from the PMMA beads, while PLA beads release more than 60% of their load, if serum is present in the release medium. The PLA system displays improved release kinetics relative to PMMA, is biodegradable, is unaltered by gas sterilization, can be used for a range of antibiotics, and can be manipulated without disintegration. These are all desirable properties for an implantable drug delivery system for the prevention or treatment of osteomyelitis.

Isosilybin B causes androgen receptor degradation in human prostate carcinoma cells via PI3K-Akt-Mdm2-mediated pathway.

The identification and development of novel nontoxic phytochemicals that target androgen and androgen receptor (AR) signaling remains a priority for prostate cancer (PCA) control. In the present study, we assessed the antiandrogenic efficacy of isosilybin B employing human PCA LNCaP (mutated AR), 22Rv1 (mutated AR) and LAPC4 (wild-type AR) cells. Isosilybin B (10-90 microM) treatment decreased the AR and prostate specific antigen (PSA) levels in LNCaP, 22Rv1 and LAPC4 cells, but not in non-neoplastic human prostate epithelial PWR-1E cells. Isosilybin B treatment also inhibited synthetic androgen R1881-induced nuclear localization of AR, PSA expression and cell growth, and caused G(1) arrest. In mechanistic studies identifying AR degradation, isosilybin B caused increased phosphorylation of Akt (Ser-473 and Thr-308) and Mdm2 (Ser-166), which was linked with AR degradation as pretreatment with PI3K inhibitor (LY294002)-restored AR level. Further, overexpression of kinase-dead Akt largely reversed isosilybin B mediated-AR degradation suggesting a critical role of Akt in AR degradation. Antibody pull-down results also indicated that isosilybin B treatment enhances the formation of complex between Akt, Mdm2 and AR, which promotes phosphorylation-dependent AR ubiquitination and its degradation by proteasome. Together, present findings identify a novel mechanism for isosilybin B-mediated anticancer effects in human PCA cells.

Homologous and heterologous protein-protein interactions of human DNA topoisomerase IIalpha.

DNA topoisomerase II (topo II; EC 5.99.1.3) is a nuclear enzyme whose DNA decatenating activity on newly replicated DNA is essential to successful cell division. Topo II catalytic activity proceeds by a concerted DNA breakage-reunion reaction coordinated between two interacting, homologous subunits. Human and yeast topo II have recently been shown to enter into heterologous protein-protein interactions and some of these interactions appear necessary for successful chromosomal segregation. In the present study, the sequences mediating homologous and heterologous protein-protein interactions have been investigated biochemically using various truncated peptides from the major alpha form of human topo II. From nonreducing gel electrophoresis and solid-phase protein-protein binding (Far Western) assays, topo II homodimerization appeared to be minimally governed by the region between amino acids 951 and 1042. However, maximal homodimerization and multimerization required sequences C-terminal to position 1042. Topo II peptides were also able to interact with 10-12 nuclear proteins from HeLa cells, termed topo II-interactive proteins or TIPs. Interestingly, small topo II peptides between residues 808 and 951 that did not homodimerize with topo II (857-1447) were nonetheless capable of binding to HeLa TIPs. These interactions were confirmed by use of topo II affinity chromatography for isolation of specific TIPs from HeLa nuclear extracts. Taken together, these data confirm that human topo II is also capable of heterologous interactions with nuclear proteins and that the region governing these interactions is distinct from, but has some overlap with, sequences directing topo II homodimerization.

Phosphorylation of DNA topoisomerase II in a human tumor cell line.

The phosphorylation of the nuclear enzyme DNA topoisomerase II was characterized from HeLa human cervical carcinoma cells labeled with 32Pi. Analysis of topoisomerase II immunoprecipitates from 32P-labeled HeLa cells indicated that phosphorylation of the enzyme occurred at serine residues. Incorporation of 32P into topoisomerase II was not due to other types of phosphomodifications such as poly(ADP-ribosylation) or covalent interactions of the enzyme with nucleic acids. The stability of topoisomerase II protein and topoisomerase II phosphorylation was also investigated in HeLa cells. Topoisomerase II protein was relatively stable, having a half-life of approximately 27 h. Phosphorylation of HeLa topoisomerase II was also remarkably stable with a T1/2 of 17 h.

Prooxidant-initiated lipid peroxidation in isolated rat hepatocytes: detection of 4-hydroxynonenal- and malondialdehyde-protein adducts.

Toxicity associated with prooxidant-mediated hepatic lipid peroxidation is postulated to originate from the interaction of the aldehydic end products of lipid peroxidation with cellular constituents. The principal alpha,beta-unsaturated aldehydic products of lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), are known to modify proteins through covalent alkylation of lysine, histidine, and cysteine amino acid residues. To detect and characterize the formation of 4-HNE- and MDA-adducted proteins during prooxidant-initiated lipid peroxidation, rabbit polyclonal antibodies were raised to 4-HNE-sulfhydryl, dinitrophenylhydrazine (DNPH)-4-HNE-sulfhydryl, and MDA-amine conjugates of keyhole limpet hemocyanin (KLH). Each antiserum displayed high antibody titers to either 4-HNE-metallothionein, DNPH-albumin, or MDA-albumin adducts when measured by ELISA. To study the formation of 4-HNE- and MDA-protein adducts during prooxidant-initiated cellular injury, isolated hepatocytes were exposed to either carbon tetrachloride or iron/ascorbate for 2 h. Indices of hepatocellular oxidative stress (i.e., cell viability and glutathione status) and lipid peroxidation (i.e., formation of 4-HNE, protein carbonyls, and MDA) were monitored continuously. Hepatocellular viability was affected moderately by carbon tetrachloride, while cellular reduced glutathione status was moderately affected by both iron/ascorbate and carbon tetrachloride. Levels of MDA and protein carbonyls increased dramatically with both prooxidants, whereas 4-HNE levels did not change significantly over the time course studied. In addition, hepatocellular proteins were immunoprecipitated with each antiserum, and aldehyde-modified immunopositive proteins were detected by immunoblotting. Prooxidant-induced increases in MDA corresponded with increases in intensity and number of MDA-adducted proteins over the time course studied. A total of 13 MDA-modified proteins (20, 25, 28, 30, 33, 38, 41, 45, 80, 82, 85, 130, and 150 kDa) were detected with the MDA-amine antiserum. Additionally, both iron/ascorbate- and carbon tetrachloride-induced formation of DNPH-derivatizable protein carbonyls corresponded quantitatively with the ability to detect specific proteins (80, 100, 130, and 150 kDa) with the DNPH-4-HNE-cysteine antiserum. Neither CCl4 nor iron/ascorbate elicited changes in 4-HNE or induced the formation of 4-HNE-modified proteins when assessed by immunoprecipitation-immunoblot analysis with the 4-HNE-sulfhydryl antiserum. In all instances detection of aldehyde-modified proteins was not associated with cell death and may be related to the function of these proteins as aldehyde-binding proteins which sequester electrophilic molecules during oxidative liver injury.