Carbonyl Reductase 1 Plays a Significant Role in Converting Doxorubicin to Cardiotoxic Doxorubicinol in Mouse Liver, but the Majority of the Doxorubicinol-Forming Activity Remains Unidentified.

Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, doxorubicinol. Identification of the enzymes responsible for doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to doxorubicinol formation were evaluated: carbonyl reductase (Cbr) 1, Cbr3, and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to doxorubicinol formation in mouse liver but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the doxorubicinol-forming activity in mouse liver cytosol but that the majority of the doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT: Earlier studies suggested carbonyl reductase (Cbr) 1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes-Cbr3 and thioredoxin reductase 1-play no role, and that the majority of the activity remains unidentified. Thus, targeting Cbr1 is necessary but not sufficient to eliminate doxorubicinol-associated cardiotoxicity; identification of the additional doxorubicinol-forming activity is an important next challenge.

Acetaminophen attenuates doxorubicin-induced cardiac fibrosis via osteopontin and GATA4 regulation: reduction of oxidant levels.

It is well documented in animal and human studies that therapy with the anti-cancer drug doxorubicin (DOX) induces fibrosis, cardiac dysfunction, and cell death. The most widely accepted mechanism of cardiac injury is through production of reactive oxygen species (ROS), which cause mitochondrial damage, sarcomere structural alterations, and altered gene expression in myocytes and fibroblasts. Here we investigated the effects of acetaminophen (APAP, N-acetyl-para-aminophenol) on DOX-induced cardiac injury and fibrosis in the presence or absence of osteopontin (OPN). H9c2 rat heart-derived embryonic myoblasts were exposed to increasing concentrations of DOX ± APAP; cell viability, oxidative stress, and OPN transcript levels were analyzed. We found a dose-dependent decrease in cell viability and a corresponding increase in intracellular oxidants at the tested concentrations of DOX. These effects were attenuated in the presence of APAP. RT-PCR analysis revealed a small increase in OPN transcript levels in response to DOX, which was suppressed by APAP. When male 10-12-week-old mice (OPN(+/+) or OPN(-/-)) were given weekly injections of DOX ± APAP for 4 weeks there was substantial cardiac fibrosis in OPN(+/+) and, to a lesser extent, in OPN(-/-) mice. In both groups, APAP decreased fibrosis to near baseline levels. Activity of the pro-survival GATA4 transcription factor was diminished by DOX in both mouse genotypes, but retained baseline activity in the presence of APAP. These effects were mediated, in part, by the ability of APAP, acting as an anti-inflammatory agent, to decrease intracellular ROS levels, consequently diminishing the injury-induced increase in OPN levels.

Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration.

Cells require ribonucleotide reductase (RNR) activity for DNA replication. In bacteria, electrons can flow from NADPH to RNR by either a thioredoxin-reductase- or a glutathione-reductase-dependent route. Yeast and plants artificially lacking thioredoxin reductases exhibit a slow-growth phenotype, suggesting glutathione-reductase-dependent routes are poor at supporting DNA replication in these organisms. We have studied proliferation of thioredoxin-reductase-1 (Txnrd1)-deficient hepatocytes in mice. During development and regeneration, normal mice and mice having Txnrd1-deficient hepatocytes exhibited similar liver growth rates. Proportions of hepatocytes that immunostained for PCNA, phosphohistone H3 or incorporated BrdU were also similar, indicating livers of either genotype had similar levels of proliferative, S and M phase hepatocytes, respectively. Replication was blocked by hydroxyurea, confirming that RNR activity was required by Txnrd1-deficient hepatocytes. Regenerative thymidine incorporation was similar in normal and Txnrd1-deficient livers, further indicating that DNA synthesis was unaffected. Using genetic chimeras in which a fluorescently marked subset of hepatocytes was Txnrd1-deficient while others were not, we found that the multigenerational contributions of both hepatocyte types to development and to liver regeneration were indistinguishable. We conclude that, in mouse hepatocytes, a Txnrd1-independent route for the supply of electrons to RNR can fully support DNA replication and normal proliferative growth.

Thioredoxin Reductase 1 Modulates Pigmentation and Photobiology of Murine Melanocytes in vivo.

Pigment-producing melanocytes overcome frequent oxidative stress in their physiological role of protecting the skin against the deleterious effects of solar UV irradiation. This is accomplished by the activity of several endogenous antioxidant systems, including the thioredoxin antioxidant system, in which thioredoxin reductase 1 (TR1) plays an important part. To determine whether TR1 contributes to the redox regulation of melanocyte homeostasis, we have generated a selective melanocytic Txnrd1-knockout mouse model (Txnrd1mel‒/‒), which exhibits a depigmentation phenotype consisting of variable amelanotic ventral spotting and reduced pigmentation on the extremities (tail tip, ears, and paws). The antioxidant role of TR1 was further probed in the presence of acute neonatal UVB irradiation, which stimulates melanocyte activation and introduces a spike in oxidative stress in the skin microenvironment. Interestingly, we observed a significant reduction in overall melanocyte count and proliferation in the absence of TR1. Furthermore, melanocytes exhibited an elevated level of UV-induced DNA damage in the form of 8-oxo-2'-deoxyguanosine after acute UVB treatment. We also saw an engagement of compensatory antioxidant mechanisms through increased nuclear localization of transcription factor NRF2. Altogether, these data indicate that melanocytic TR1 positively regulates melanocyte homeostasis and pigmentation during development and protects against UVB-induced DNA damage and oxidative stress.

Effect of thioredoxin deletion and p53 cysteine replacement on human p53 activity in wild-type and thioredoxin reductase null yeast.

Reporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. To investigate the role of thioredoxin in controlling p53 activity, the level of reporter gene transactivation by p53 was determined in yeast lacking the TRX1 and TRX2 genes encoding cytosolic thioredoxin. Surprisingly, p53 activity was unimpaired in yeast lacking thioredoxin. Subsequent analyses showed that thioredoxin deletion suppressed the inhibitory effect of thioredoxin reductase deletion, suggesting that accumulation of oxidized thioredoxin in mutant yeast was necessary for p53 inhibition. Purified human thioredoxin and p53 interacted in vitro (Kd = 0.9 microM thioredoxin). To test the idea that dithio-disulfide exchange reactions between p53 and thioredoxin were responsible for p53 inhibition in mutant yeast, each p53 cysteine was changed to serine, and the effect of the substitution on p53 activity in TRR1 and Deltatrr1 yeast was determined. Substitutions at Zn-coordinating cysteines C176, C238, or C242 resulted in p53 inactivation. Unexpectedly, substitution at cysteine C275 also inactivated p53, which was the first evidence for a non-zinc-coordinating cysteine being essential for p53 function. Cysteine substitutions at six positions (C124, C135, C141, C182, C229, and C277) neither inactivated p53 nor relieved the requirement for thioredoxin reductase. Furthermore, no tested combination of these six cysteine substitutions relieved thioredoxin reductase dependence. The results suggested that p53 dependence on thioredoxin reductase either was indirect, perhaps mediated by an upstream activator of p53, or was due to oxidation of one or more of the four essential cysteines.

Cholestatic liver disease results increased production of reactive aldehydes and an atypical periportal hepatic antioxidant response.

Cholangiopathies such as primary sclerosing cholangitis (PSC) are chronic liver diseases characterized by increased cholestasis, biliary inflammation and oxidative stress. The objective of this study was to elucidate the impact of cholestatic injury on oxidative stress-related factors. Using hepatic tissue and whole cell liver extracts (LE) isolated from 11-week old C57BL/6J (WT) and Mdr2KO mice, inflammation and oxidative stress was assessed. Concurrently, specific targets of carbonylation were assessed in LE prepared from murine groups as well as from normal and human patients with end-stage PSC. Identified carbonylated proteins were further evaluated using bioinformatics analyses. Picrosirius red staining revealed extensive fibrosis in Mdr2KO liver, and fibrosis colocalized with increased periportal inflammatory cells and both acrolein and 4-HNE staining. Western blot analysis revealed elevated periportal expression of antioxidant proteins Cbr3, GSTµ, Prdx5, TrxR1 and HO-1 but not GCLC, GSTπ or catalase in the Mdr2KO group when compared to WT. From immunohistochemical analysis, increased periportal reactive aldehyde production colocalized with elevated staining of Cbr3, GSTµ and TrxR1 but surprisingly not with Nrf2. Mass spectrometric analysis revealed an increase in carbonylated proteins in the Mdr2KO and PSC groups compared to respective controls. Gene ontology and KEGG pathway analysis of carbonylated proteins revealed a propensity for increased carbonylation of proteins broadly involved in metabolic processes as well more specifically in Rab-mediated signal transduction, lysosomes and the large ribosomal subunit in human PSC. Western blot analysis of Rab-GTPase expression revealed no significant differences in Mdr2KO mice when compared to WT livers. In contrast, PSC tissue exhibited decreased levels of Rabs 4, 5 and increased abundance of Rabs 6 and 9a protein. Results herein reveal that cholestasis induces stage-dependent increases in periportal oxidative stress responses and protein carbonylation, potentially contributing to pathogenesis in Mdr2KO. Furthermore, during early stage cholestasis, there is cell-specific upregulation of some but not all, antioxidant proteins.

Acetaminophen is cardioprotective against H2O2-induced injury in vivo.

Here we report our ongoing investigation of the cardiovascular effects of acetaminophen, with emphasis on oxidation-induced canine myocardial dysfunction. The objective of the current study was to investigate whether acetaminophen could attenuate exogenous H(2)O(2)-mediated myocardial dysfunction in vivo. Respiratory, metabolic, and hemodynamic indices such as left ventricular function (LVDP and +/-dP/dt(max)), and percent ectopy were measured in anesthetized, open-chest dogs during intravenous administration of 0.88 mM, 2.2 mM, 6.6 mM H(2)O(2). Following 6.6 mM H(2)O(2), tissue from the left ventricle was harvested for electron microscopy. Left ventricular function did not vary significantly between vehicle and acetaminophen groups under baseline conditions. Acetaminophen-treated dogs regained a significantly greater fraction of baseline function after high concentrations of H(2)O(2) than vehicle-treated dogs. Moreover, the incidence of H(2)O(2)-induced ventricular arrhythmias was significantly reduced in the acetaminophen-treated group. Percent ectopy following 6.6 mM concentrations of H(2)O(2) was 1 +/- 0.3 vs. 0.3 +/- 0.1 (P < 0.05) for vehicle- and acetaminophen-treated dogs, respectively. Additionally, electron micrograph images of left ventricular tissue confirmed preservation of tissue ultrastructure in acetaminophen-treated hearts when compared to vehicle. We conclude that, in the canine myocardium, acetaminophen is both functionally cardioprotective and antiarrhythmic against H(2)O(2)-induced oxidative injury.

Elevated Nrf-2 responses are insufficient to mitigate protein carbonylation in hepatospecific PTEN deletion mice.

OBJECTIVE: In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/ß unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model. METHODS: Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTENLKO) or control littermates were fed a standard chow diet for 45-55 weeks followed by analysis for liver injury, oxidative stress and inflammation. RESULTS: Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTENLKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTµ, GSTπ and CBR1/3, but not HO-1, were elevated in PTENLKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTENLKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTENLKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-S-transferases (GST) M6, O1, and O2. CONCLUSIONS: Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation.

Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome.

Thioredoxin reductases (Txnrd) maintain intracellular redox homeostasis in most organisms. Metazoan Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1-/- cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated that primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd-/- and txnrd+/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however, mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells.

Antiarrhythmic properties of acetaminophen in the dog.

Mongrel dogs bred for research and weighing 25 +/- 3 kg were used to test the hypothesis that acetaminophen has antiar-rhythmic properties. Only ventricular arrhythmias defined by the Lambeth Conventions were investigated. Dogs were exposed either to 60 mins of regional myocardial ischemia followed by 180 mins of reperfusion (n = 14) or were administered a high dose of ouabain (n = 14). Both groups of 14 dogs were further divided into vehicle and acetaminophen treatment groups (n = 7 in each). During selected 10-min intervals, we recorded the numbers of ventricular premature beats, ventricular salvos, ventricular bigeminy, ventricular tachycardia (nonsustained and sustained), and we recorded the heart rate, systemic arterial blood pressure, and left ventricular function. Neither heart rate nor the number of ventricular arrhythmias differed significantly under baseline conditions. Conversely, the combined average number of ventricular ectopic beats during ischemia and reperfusion was significantly less in the presence of acetaminophen (28 +/- 4 vs. 6 +/- 1; P < 0.05). Similarly, percent ectopy during reperfusion in vehicle- and acetaminophen-treated dogs was 1.4 +/- 0.4 and 0.4 +/- 0.2, respectively (P < 0.05). The number of all ventricular ectopic beats except ventricular salvos was also significantly reduced in the presence of acetaminophen. Similar results were obtained with ouabain. Our results reveal that systemic administration of a therapeutic dose of acetaminophen has previously unreported antiarrhythmic effects in the dog.

Experimentally-induced ventricular arrhythmias.

Hypoxia and reoxygenation, ischemia and reperfusion, catecholamine infusion, ouabain, sodium pentobarbital and caffeine, can all be used experimentally to induce ventricular arrhythmias. According to the Lambeth Convention guidelines our experimentally-induced ventricular arrhythmias include but are not limited to: ventricular premature beats (VPB), ventricular salvos (VS), ventricular bigeminy (VB), nonsustained ventricular tachycardia (VTn), sustained ventricular tachycardia (VTs) and ventricular fibrillation (VF, or if the heart is not defibrillated, sudden cardiac death). We have studied these arrhythmias in the absence and presence of adenosine deaminase, methyl xanthines, and more recently, acetaminophen. Our laboratory was the first to discover the anti-arrhythmic properties of acetaminophen an analgesic used in Western medicine for more than 100 years before our publication. We have also identified other cardioprotective properties of acetaminophen, and have begun to work out some of the cellular/molecular mechanisms. For example, we know that acetaminophen protects hypoxic/ischemic cardiac mitochondria, in part, by sustaining function of the mitochondrial permeability transition pore (MPTP, a protein involved in regulating mitochondrial pH). Acetaminophen also attenuates the actions of matrix metalloproteinases that can be harmful to myocardial contractile proteins. Of course, like all science, more work is needed to expand on these and related topics.

Serum thioredoxin reductase is highly increased in mice with hepatocellular carcinoma and its activity is restrained by several mechanisms.

Increased thioredoxin reductase (TrxR) levels in serum were recently identified as possible prognostic markers for human prostate cancer or hepatocellular carcinoma. We had earlier shown that serum levels of TrxR protein are very low in healthy mice, but can in close correlation to alanine aminotransferase (ALT) increase more than 200-fold upon chemically induced liver damage. We also found that enzymatic TrxR activity in serum is counteracted by a yet unidentified oxidase activity in serum. In the present study we found that mice carrying H22 hepatocellular carcinoma tumors present highly increased levels of TrxR in serum, similarly to that reported in human patients. In this case ALT levels did not parallel those of TrxR. We also discovered here that the TrxR-antagonistic oxidase activity in serum is due to the presence of quiescin Q6 sulfhydryl oxidase 1 (QSOX1). We furthermore found that the chemotherapeutic agents cisplatin or auranofin, when given systemically to H22 tumor bearing mice, can further inhibit TrxR activities in serum. The TrxR serum activity was also inhibited by endogenous electrophilic inhibitors, found to increase in tumor-bearing mice and to include protoporphyrin IX (PpIX) and 4-hydroxynonenal (HNE). Thus, hepatocellular carcinoma triggers high levels of serum TrxR that are not paralleled by ALT, and TrxR enzyme activity in serum is counteracted by several different mechanisms. The physiological role of TrxR in serum, if any, as well as its potential value as a prognostic marker for tumor progression, needs to be studied further.

An old drug with a new purpose: cardiovascular actions of acetaminophen (paracetamol).

For over 50 years, acetaminophen (paracetamol) has been a staple in industrialized and non-industrialized countries for the treatment of pain and fever. Although its precise mechanisms of action are not known, the drug generates dose-dependent reduction in circulating prostaglandins, inhibits myeloperoxidase and the oxidation of lipoproteins, and appears to confer cardioprotection by blocking the effects of hydroxyl radical, peroxynitrite, and hydrogen peroxide. The drug might inhibit cyclooxygenase, although its ultimate target(s) is (are) still unclear. Sadly, since most investigations of acetaminophen have focused on its analgesic/antipyretic properties and hepatotoxicity, the effects of the drug on other mammalian organ systems, including the heart and circulation, have been ignored. Recently, work in our laboratory has shown acetaminophen to have a protective role in the injured mammalian myocardium. The cardioprotection was first observed in isolated, perfused guinea pig hearts subjected to ischemia-reperfusion injury. Hearts pretreated with acetaminophen recovered greater ventricular function and exhibited improved myofibrillar ultrastructure when compared to vehicle-treated hearts. More recent in vitro investigations have suggested protective roles for acetaminophen in barbiturate-induced arrhythmogenesis and myocardial hypoxia-reoxygenation injury. We have also extended our work to the in vivo arena. There, we found that acetaminophen reduced infarct size in dogs exposed to 60 minutes regional myocardial ischemia and 180 minutes reperfusion. We invite and encourage readers of this review to repeat/duplicate our experiments. Such work is needed to either challenge or support our findings. Further, more clinically-relevant work depends on these basic and related translational experiments.

Metabolism of doxorubicin to the cardiotoxic metabolite doxorubicinol is increased in a mouse model of chronic glutathione deficiency: A potential role for carbonyl reductase 3.

Doxorubicin is highly effective at inducing DNA double-strand breaks in rapidly dividing cells, which has led to it being a widely used cancer chemotherapeutic. However, clinical administration of doxorubicin is limited by off-target cardiotoxicity, which is thought to be mediated by doxorubicinol, the primary alcohol metabolite of doxorubicin. Carbonyl reductase 1 (CBR1), a well-characterized monomeric enzyme present at high basal levels in the liver, is known to exhibit activity toward doxorubicin. Little is known about a closely related enzyme, carbonyl reductase 3 (CBR3), which is present in the liver at low basal levels but is highly inducible by the transcription factor Nrf2. Genetic polymorphisms in CBR3, but not CBR1, are associated with differential cardiac outcomes in doxorubicin treated pediatric patients. Cbr3 mRNA and CBR3 protein are highly expressed in the livers of Gclm-/- mice (a mouse model of glutathione deficiency) relative to wild type mice. In the present study, we first investigated the ability of CBR3 to metabolize doxorubicin. Incubations of doxorubicin and purified recombinant murine CBR3 (mCBR3) were analyzed for doxorubicinol formation using HPLC, revealing for the first time that doxorubicin is a substrate of mCBR3. Moreover, hepatocytes from Gclm-/- mice produced more doxorubicinol than Gclm+/+ hepatocytes. In addition, differentiated rat myoblasts (C2C12 cells) co-cultured with primary Gclm-/- murine hepatocytes were more sensitive to doxorubicin-induced cytostasis/cytotoxicity than incubations with Gclm+/+ hepatocytes. Our results indicate a potentially important role for CBR3 in doxorubicin-induced cardiotoxicity. Because there is likely to be variability in hepatic CBR3 activity in humans (due to either genetic or epigenetic influences on its expression), these data also suggest that inhibition of CBR3 may provide protection from doxorubicinol cardiotoxicity.

Acetaminophen in the hypoxic and reoxygenated guinea pig myocardium.

We investigated the effects of 0.35-mM acetaminophen and its vehicle on isolated, perfused guinea pig hearts made hypoxic and subsequently reoxygenated. Hearts were allowed 30 min postinstrumentation to reach baseline, steady-state values, and then were exposed to 6 min of hypoxia (5% O(2), 5% CO(2), balance N(2)) followed by 36 min of reoxygenation (95% O(2), 5% CO(2)). We recorded hemodynamic, metabolic, and mechanical data in addition to assessing ultrastructure and the capacity of coronary venous effluent to reduce reactive oxygen species. We found that acetaminophen-treated hearts retained a greater fraction of mechanical function during hypoxia and reoxygenation. For example, the average percentage change from baseline of left ventricular developed pressure in acetaminophen- and vehicle-treated hearts at 6 min reoxygenation was 9 +/- 2% and -8 +/- 5% (P < 0.05), respectively. In addition, electron micrographs revealed greater preservation of myofibrillar ultrastructure in acetaminophen-treated hearts. Biochemical analyses revealed the potential of coronary effluent from acetaminophen-treated hearts to significantly neutralize peroxynitrite-dependent chemiluminescence in all recorded time periods. During early reoxygenation, the percentage inhibition of peroxynitrite-mediated chemiluminescence was 56 +/- 10% in vehicle-treated hearts and 99 +/- 1% in acetaminophen-treated hearts (P < 0.05). We conclude that acetaminophen has previously unreported cardioprotective properties in the nonischemic, hypoxic, and reoxygenated myocardium mediated through the reduction of reactive oxygen species.

cDNA-directed expression of a functional zebrafish CYP1A in yeast.

A cytochrome P450 1A (CYP1A) cDNA was isolated from an adult zebrafish (Danio rerio) library. The 2580-bp clone (GenBank Accession No. AF210727) contained a 62-bp 5'-unstranslated region (UTR), 1557-bp coding region and 962-bp 3'-UTR. The deduced 519-residue protein (calculated molecular weight 58,556, pI = 7.58) shared 74% identity with rainbow trout CYP1A and 57 and 54% identities with mouse and human CYP1A1s, respectively. The zebrafish CYP1A protein coding region was cloned into the pDONR201 entry vector and then transferred to a yeast expression vector pYES-DEST52. Expression of zebrafish CYP1A in Saccharomyces cerevisiae transformants was induced by galactose to a maximum level of 493 pmol CYP1A per mg microsomal protein or about 8 nmol/l of culture. Recombinant CYP1A protein expressed in yeast was mainly in the denatured P420 form under normal microsomal preparation conditions but when the oxygen concentration was reduced in the buffer by degassing and the yeast cells were maintained at less than 10 degrees C, the integrity of the CYP1A was preserved and it exhibited a characteristic reduced CO-difference spectrum maximum at 448 nm. The recombinant zebrafish CYP1A demonstrated 7-ethoxyresorufin O-deethylase (EROD) activity with an apparent Km (Km(app)) and Vmax values at 30 degrees C of 0.31 +/- 0.04 microM and 0.70 +/- 0.10 nmol/min/nmol CYP, respectively. The recombinant protein also metabolized benzo(a)pyrene with a Km(app) and Vmax values of 5.34 +/- 0.58 microM and 1.16 +/- 0.13 nmol/min/nmol CYP, respectively. These results show the recombinant expression of a functional zebrafish CYP in yeast and validated yeast as a host for heterologous expression of zebrafish CYP1A and potentially for other zebrafish CYPs.

Endothelin-1 is a transcriptional target of p53 in epidermal keratinocytes and regulates ultraviolet-induced melanocyte homeostasis.

Keratinocytes contribute to melanocyte activity by influencing their microenvironment, in part, through secretion of paracrine factors. Here, we discovered that p53 directly regulates Edn1 expression in epidermal keratinocytes and controls UV-induced melanocyte homeostasis. Selective ablation of endothelin-1 (EDN1) in murine epidermis (EDN1(ep-/-) ) does not alter melanocyte homeostasis in newborn skin but decreases dermal melanocytes in adult skin. Results showed that keratinocytic EDN1 in a non-cell autonomous manner controls melanocyte proliferation, migration, DNA damage, and apoptosis after ultraviolet B (UVB) irradiation. Expression of other keratinocyte-derived paracrine factors did not compensate for the loss of EDN1. Topical treatment with EDN1 receptor (EDNRB) antagonist BQ788 abrogated UV-induced melanocyte activation and recapitulated the phenotype seen in EDN1(ep-/-) mice. Altogether, the present studies establish an essential role of EDN1 in epidermal keratinocytes to mediate UV-induced melanocyte homeostasis in vivo.

Osteopontin expression during early cerebral ischemia-reperfusion in rats: enhanced expression in the right cortex is suppressed by acetaminophen.

Osteopontin (OPN) is a pleiotropic protein implicated in various inflammatory responses including ischemia-reperfusion (I-R) injury. Two distinct forms of the protein have been identified: an extensively studied secreted form (sOPN) and a less-well-known intracellular form (iOPN). Studies have shown that increased OPN expression parallels the time course of macrophage infiltration into injured tissue, a late event in the development of cerebral infarcts. sOPN has been suggested to promote remodeling of the extracellular matrix in the brain; the function of iOPN may be to facilitate certain signal transduction processes. Here, we studied OPN expression in adult male Sprague-Dawley rats subjected to global forebrain I-R injury. We found iOPN in the cytoplasm of both cortices and the hippocampus, but unexpectedly only the right cortex exhibited a marked increase in the iOPN level after 45 min of reperfusion. Acetaminophen, a drug recently shown to decrease apoptotic incidence, caspase-9 activation, and mitochondrial dysfunction during global I-R, significantly inhibited the increase in iOPN protein in the right cortex, suggesting a role for iOPN in the response to I-R injury in the right cortex.

Acetaminophen reduces mitochondrial dysfunction during early cerebral postischemic reperfusion in rats.

Acetaminophen, a popular analgesic and antipyretic, has been found to be effective against neuronal cell death in in vivo and in vitro models of neurological disorders. Acute neuronal death has been attributed to loss of mitochondrial permeability transition coupled with mitochondrial dysfunction. The potential impact of acetaminophen on acute injury from cerebral ischemia-reperfusion has not been studied. We investigated the effects of acetaminophen on cerebral ischemia-reperfusion-induced injury using a transient global forebrain ischemia model. Male Sprague-Dawley rats received 15mg/kg of acetaminophen intravenously during ischemia induced by hypovolemic hypotension and bilateral common carotid arterial occlusion, which was followed by reperfusion. Acetaminophen reduced tissue damage, degree of mitochondrial swelling, and loss of mitochondrial membrane potential. Acetaminophen maintained mitochondrial cytochrome c content and reduced activation of caspase-9 and incidence of apoptosis. Our data show that acetaminophen reduces apoptosis via a mitochondrial-mediated mechanism in an in vivo model of cerebral ischemia-reperfusion. These findings suggest a novel role for acetaminophen as a potential stroke therapeutic.