Figure 1. Immunization with TGF-β1 kinoids elicits TGF-β1 neutralizing IgG Abs. (A) Male BALB/c mice were i.p. injected with the TGF-β1 kinoids (50 μg in 0.2 mL), keyhole limpet hemocyanin (KLH, 50 μg in 0.2 mL) or an equal volume of phosphate-buffered saline, 2 weeks apart a total of four times. Indirect ELISA with recombinant human TGF-β1-coated (20 ng/well) plates showed that immunization with TGF-β1 kinoids stimulated high-titer and long-term anti–TGF-β1 IgG Abs. (B) Western blotting showed that four immunizations with TGF-β1 kinoids elicited the production of specific IgG Abs that could bind to membrane-bound recombinant human TGF-β1. A total of 5 μg of prokaryotically expressed human TGF-β1 (prTGF-β1) on each lane was applied to SDS-PAGE. The sera from the mice after 4 injections ofTGF-β1 kinoids, KLH or PBS were used as the primary antibodies (1:800). (C) The antisera produced byTGF-β1 kinoid immunization showed dose-dependent neutralization activities on the TGF-β1-induced growth- inhibition on mink lung epithelial cells (Mv1Lu). After a 48-hour starvation in serum-free medium, Mv1Lu cells in 96-well plates were treated with TGF-β1 (2.5 ng/mL) and the various dilutions of the sera from the TGF-β1 kinoid-immunized or KLH-injected mice. Forty-eight hours post-treatment, BrdU incorporation in the cells was determined by quantitative ELISA. The incorporation of BrdU in the untreated Mv1Lu cells was set to 1. The results shown represent the mean ± the standard error of the mean (SEM) of three independent experiments, each of which was performed in triplicate wells. The error bars indicate SEM.
Cholangiocyte accumulation is a poor predictor for hepaticfibrosis. Our study demonstrated that TGF-b1-treated 603B cells were more resistant to TNF-a/SC-514-induced apoptosis. Snail1 knockdown significantly attenuated the protective effects ofTGF- b1 on 603B cells, suggesting that TGF-b1-induced EMT-like alterations might contribute to cholangiocyte accumulation during liver fibrosis via suppressing apoptotic cell death. Increasing evidence supports that induction of EMT is generally associated with reduced apoptosis and the molecular mechanisms are not fully understood. We found a modest downregulation of multiple pro-apoptotic genes, including Bax, Bid, Bim, Pten, and Puma, in 603B cells after continuous TGF-b1 stimulation. Snail1 knock- down abolished down-regulation of Bid, Bim and Puma inducedbyTGF-b1 exposure, further supporting the involvement EMT- associated signaling in the development of apoptotic resistance inTGF-b1-treated cells. Genome-wide ChIP analysis suggested that Snail1 can bind the promoters of many apoptosis-associated genes when it was overexpressed in ovarian cancer cells . Whether TGF-b1-induced Snail1 can directly silence these pro-apoptotic genes merits further investigation. In addition, the regulatory effects of Snail1 on apoptosis might not limit to silencing pro- apoptotic genes. A recent study suggested that snail1 knockdown prevented the upregulation of anti-apoptotic molecules, Bcl-xL and Mcl-1, in mouse hepatocytes inducedbyTGF-b1 . Obviously, many anti-apoptotic and pro-apoptotic molecules and activation of multiple EMT-associated signal pathways are involved. Targeting accumulated proliferative cholangiocytes has been shown to attenuate liver fibrosisinmice . A compre- hensive evaluation of the role for EMT signaling in cholangiocyte apoptotic resistance during liver fibrosis may help to identify new targets for therapeutic intervention. It will be also of interest to extend these studies to other cholangiocyte cell lines, as well as to determine the role ofTGF-b1-induced cholangiocyte EMT in ECM production during liver fibrosisin vivo.
Undernourished mice infected (UI) submitted to low and long-lasting infections by Schistosoma mansoni are unable to develop the hepatic periportal fibrosis that is equivalent to Symmers’ fibrosisin humans. In this report, the effects of the host’s nutritional status on parasite (worm load, egg viability and maturation) and host (growth curves, biology, collagen synthesis and characteristics of the immunological response) were studied and these are considered as interdependent factors influencing the amount and distribution of fibrous tissue inhepatic periovular granulomas and portal spaces. The nutritional status of the host influenced the low body weight and low parasite burden detected in UI mice as well as the number, viability and maturation of released eggs. The reduced oviposition and increased number of degenerated or dead eggs were associated with low protein synthesis detected in deficient hosts, which likely induced the observed decrease in transformation growth factor (TGF)-β1 and liver collagen. Despite the reduced number of mature eggs in UI mice, the activation ofTGF-β1 and hepatic stellate cells occurred regardless of the unviability of most miracidia, due to stimulation by fibrogenic proteins and eggshell glycoproteins. However, changes in the repair mechanisms influenced by the nutritional status in deficient animals may account for the decreased liver collagen detected in the present study.
Reactive oxygen species (ROS) are generated by various liver injuries such as alcohol abuse, hepatitis virus infection and chronic cholestasis and contribute to hepatic fibrogenesis . ROS stimulates the production of the Collagen I, acting as an intracellular signaling mediator of the fibrogenic action ofTGF-β1 . The multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) enzyme complexes and the mitochondrial respiratory pathway are the two major producers of endogenous ROS . NOX play a central role in liver fibrogenesis. Among the seven members of the NOX family, NOX1 is structurally and func- tionally similar to NOX2, the classic NOX that generates the oxidative burst in neutrophil kill- ing. Studies by us and others have shown that NOX1 and NOX2 are expressed in HSCs and deficiencies of NOX1 or NOX2 decrease liver inflammation and fibrosisin the carbon tetra- chloride (CCL4) and bile duct ligation (BDL) models [5, 9]. Angiotensin II (Ang II) also induces NOX1 to promote HSCs proliferation and aggravate liver fibrosis [5, 9]. In contrast, NOX4, a nonphagocytic NOX homolog is expressed in the liver, and is different from the other NOX isoforms because it does not require the recruitment of cytosolic structural subunits to form the active enzyme to produce ROS [10, 11]. NOX4 is critical in lung and kidney fibrosisby activating and transforming of myofibroblasts [12, 13]. In the liver, NOX4 is expressed in hepatocytes, stellate cells, and endothelial cells . NOX4 has been found to be upregulated in hepatitis virus C, and to contribute to the formation of ROS, most likely via TGF-β1 induction . The role of NOX4 in liver injury and fibrosis has only been assessed in the BDL model using NOX4 deficient mice . A concern about these previous studies is that they were per- formed by breeding homozygous knock-out mice compared to wild type strain matched con- trol mice, which could result in artifact genetic drift in the two groups.
We previously described a selective bile duct ligation model to elucidate the process ofhepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (a-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-b1, molecules known to be involved inhepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosisin this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT- PCR and in real-time quantitative PCR (qRT-PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, a-SMA and TGF-b1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and inhepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT-PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased a-SMA and TGF-b1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased a-SMA 60 days after surgery. The qRT-PCR technique was more sensitive to expression changes than the semiquantitative method.
Intermediate stage - This stage of repair occurred about the same time for both rats and mice (40-45 days after inoculation) and was characterized by the subsiding of the reactive hepatitis. In rats live worms disappeared from the focal leions, being replaced by partially calcified worm debris (Fig. 1A), and collections of immature eggs. A repair tissue formed by fibroblast and blood vessel pro- liferation and macrophage infiltration later replaced the necrotic areas, but packed collections of eggs were still present, although in the absence of worm debris, espe- cially inmice. The signs of non-specific hepatitis seen during the acute stage, completely disappeared in rats, but left a variable degree of peri-sinusoidal fibrosisinmice (Fig. 2C, D). However, septal fibrosis formation was a constant and prominent change seen in rats, although absent inmice. The early septa were thin and cellular, and they connected portal spaces to neighboring portal spaces sometimes forming polyhedral figures with the central area occupied by central veins, mimicking the normal his- tology of the pig liver. The septa were formed by parallel collagen fibers, fusiform cells (fibroblasts) and blood ves- sels (Fig. 1C, D). Hepatic cells present at the vicinity of the septum sometimes showed apoptotic changes, even- tually dropping out and being incorporated into the sep- tal structure. Some of them were seen to contain PAS- positive, diastase-resistant material, and a brownish, iron- containing pigment.
The carrageenan-induced paw edema model has frequently been used to evaluate the antiedematogenic effect of most anti-inlammatory drugs in routine clinical use (Carvalho et al., 1999). Reports describe that several mediators are released through the injection of carra- geenan in the rat’s paw; while histamine, serotonin and bradykinin are released in the initial phase (0-1h), in the later phase (1-6 h) there is an increase in the production levels of prostaglandins (PGs) through the activation of cyclooxygenase-2 (COX-2) and release of nitric oxide (NO) (Silva et al., 2005).
In 2005, sorafenib became the first FDA-approved oral agent for the treatment of patients with advanced hepatocellular and renal cell carcinomas . Previous reports largely focused on the role of sorafenib in tumors and apoptosis via blocking receptor tyrosine kinases. In this study, we discovered a novel action of sorafenib, namely, it significantly suppressed TGF-β-in- duced EMT and apoptosis in NRK-52E cells and attenuated renal fibrosisin a UUO model. The available evidence has shown that TGF-β is the major mediator of progressive renal fibro- sis, largely via the Smad-dependent pathway. However, the exact molecular mechanisms un- derlying the link between TGF-β and disease progression in kidney fibrosis have remained
Transforming growth factor (TGF)-bl influences a wide variety of important cellular activities and is secreted by a diverse range of cells that include immune cells localizing to inflammatory sites . Importantly, TGF-b1 can stimulate osteoblast proliferation and regulate osteoclast functions, such as the production and secretion of osteoclast Wnt10b, and could contribute to coupling . Therefore, TGF-b1 has potential as a promising candidate for the treatment of periodontal diseases. However, recent studies have revealed that TGF-b1 is a pivotal modulator of connective tissue regeneration and bone remodeling . Here, TGF-b1 induces differentiation and proliferation of osteoblasts and their precursors, with the precise response dependent on the cell phenotype and stage of maturity [8–10]. TGF-b1 also increases alkaline phosphatase (ALP) activity in murine bone marrow stromal cells . Although TGF-b1 promotes osteoblast differentiation and bone formation [12–15], it inhibits osteogenesis by various mechanisms depending on its concentration, the cell density, and the
the proinﬂammatory cytokines, TNF- a , IL-6, and MCP-1, which was consistent with the results found earlier with the paw edema in- ﬂammatory model . According to other investigators [5,33], during the early phase of sepsis, TNF- a and IL-1 are the main ini- tiators of septic responses, released predominantly from activated macrophages within 30 min after the infectious stimulus. Once in the systemic circulation, these cytokines stimulate different target cells, such as endothelial cells, which upregulate expression of adhesion molecules and enhance integrin adhesiveness to neu- trophils, leading to excessive extravasation into tissues. TNF- a and IL-1 are also responsible for amplifying later proinﬂammatory cytokine production (e.g., IL-6 and IL-8). Elevated IL-6 blood levels are associated with mediation of the acute phase response in sepsis that involves fever, leukocytosis, and multiple organ failure. Moreover, IL-6 is an indicator of sepsis severity because it can be found at high levels for an extended period.
tions, in the control or the UR. The bifur- cated conditioning response (CR) evoked in the PK neuron by this mechanical stimulus (2) was eliminated by sectioning the left third nerve (Figure 1). An electrical stimulus was delivered to the PK neuron as the SCS (Figure 1). An action potential was evoked in the PK neuron by a threshold (4 nA) SCS in Figure 3-3 of the second article (2). In order not to confuse this action potential with the UR or with the alpha-response (8), the SCS in the present article must not evoke any response in the PK neuron. Such an SCS was found by breaking the long electrical pulse (50.0 ms) used in the last article (2) into a train of the same duration (50.0 ms) but consisting of short (0.1 ms) electrical pulses (SCS, Figure 1 and SCS, Figure 4-3). The amplitude of the SCS was maintained at the threshold value of 4 nA in the present study although it did not evoke any response in the PK neuron or in the M-2 neuron even by a superthreshold amplitude of more than 10.0 nA in either polarity. Further advan- tages of using this new SCS are described in the next paragraph. The US parameters, i.e., peak numbers and amplitude, must be main- tained constant throughout the duration of the control experiment. The first parameter, the UR peak number, was always constant as 1.0 ± 0.0 (Figure 1) after sectioning the left third nerve (2). This number changed only in experiments ofinduced modifications. The other parameter, the amplitude (AMP) of these UR peaks, was not always constant when evoked by repeated US in constant intertrial intervals (ITI). A long ITI which evoked nearly constant UR amplitude was used in control experiments (Figure 2). As these UR amplitudes were never exactly con- stant (see Results), they were measured by three parameters, the average amplitudes, , the sum of all amplitudes, Σ AMP , and the highest peak amplitude (V) ever recorded in 12 min by repeated US in constant ITI. The reason for limiting these experiments to 12-min sessions but not any longer was the
Progressive removal of excess fibrous tissue, revealed by morphologic and/or biochemical evidences of collagen degradation, has long been recognized to occur in both physiological and pathological conditions (15, 17). The morphology of collagen degradation markedly differs when recent and old fibroses are in the process of being removed, as observed in experimental hepatic schistosomiasis ofmice (3, 4) and during the involution of the Seyle´s inflammatory pouch in rats (11). These differences, also observed on hepatosplenic schistosomiasis of man (1, 5, 12), have led to the suggestion that the mechanisms operating in acute and chronic collagen degradation may also differ, since morphology and function are closely related. Most experimental models used for the study of extra-cellular matrix degradation (metamorphosis of the tadpole tail, the carrageenin granuloma, involution of the pregnant rat uterus, treated acute schistosomiasis, etc) are representatives of the acute type of degradation. They reveal the main differential ultrastructural features mentioned above as typical of acute degradation (16, 13). To further explore on the correlation between time offibrosis installation and rapidity offibrosis degradation on one side, and morphological findings on another, an investigation was made on early and late stages of the carbon tetrachloride-inducedhepaticfibrosisin rats, after two different periods of drug administration, followed by three periods of observation after drug withdrawal. This study was also considered to, by exploring the subject of acute versus chronic collagen degradation in a well known experi- mental model ofhepaticfibrosis, contribute to the model itself, and stimulate further research on one important topic ofhepatic pathology.
This study was carried out using micein strict accordance with the national, european (EU Directives 86/609/EEC) and inter- national guidelines in use at the Universite´ Libre de Bruxelles. All procedures were reviewed and approved by the ethics committee (Commission d’Ethique du Bien-Etre Animal, CEBEA) of the Universite´ Libre de Bruxelles (Permit Number: 338N, 146N and 341N). All efforts were made to minimize suffering: mice were placed in a ventilated room with all appropriate hygiene and feeding conditions throughout the experiments. Housing, inocu- lation, data collection, and euthanasia procedures complied with National Institutes of Health guidelines, and the experimental protocol was approved by the Bioethics Committee of the University of Lie`ge. Mice were monitored twice daily and in the case of a weight loss exceeding 30% of the body weight or clear signs of animal suffering, mice were euthanized by cervical dislocation and integrated in the mortality curve data.
two-fold dilutions of the sera were made in culture medium containing 2 µg/ml actino- mycin-D (Calbiochem-Boehring Corp., La Jolla, CA). One hundred µl of each dilution was added to wells containing a confluent monolayer of L929B cells. After 18-20 h of incubation, plates were fixed and stained with 0.2% crystal violet in 2% ethanol solu- tion. Plates were washed and analyzed with an ELISA plate reader (Dynatech Laborato- ries, Chantilly, VA) at 570 nm. TNF titer (U/ ml) was defined as the inverse of the dilution that caused 50% destruction of the mono- layer. Serum TNF- α levels were also evalu- ated by ELISA (see below), and the correla- tion coefficient between the TNF- α levels measured by ELISA or bioassay was r = 0.93 and P = 0.0001.
anomaly [16, 50] although our current finding suggests that ADH is capable of attenuating tunicamycin-induced rises in ER stress, ROS, and autophagy. Earlier evidence suggested that Class I and IV ADH may reduce endogenous aldehyde and 4-HNE [18, 19]. Our experimental findings revealed that ADH attenuated tunicamycin-induced ER stress in concert with the changes in ROS production. Although the precise mechanism of action behind ADH-offered protection against tunicamycin-induced autophagy induction is unknown at this time, two mechanisms may be considered. First, ER stress is known to impair cardiac function through inducing autophagy. It was also proved by our in vitro study that inducing autophagy by inhi- bition of Akt and mTOR abolished the ADH-offered protection against tunicamycin. Second, restored PTEN signaling may play a role in ADH-offered inhibition of autophagy. Given that ROS and its product 4-HNE may suppress PTEN phosphorylation, ADH may restore PTEN phosphorylation level by suppression of ROS. Restored PTEN phosphorylation leads to the activation of the PTEN downstream signaling Akt-mTOR cascade, leading to suppressed autophagy. Inhibition of Akt or mTOR diminished ADH-offered protective effect against tuni- camycin according to the in vitro study. As mentioned earlier, ERK/JNK signaling pathway is unlikely to be involved in ADH-offered protection in autophagy, and possibly mechanical ben- efit under ER stress. Taken together, these findings favored an important role of PTEN-Akt- mTOR pathway in ADH transgene-offered protection of tunicamycin-induced excessive autophagy.
The elucidation of the pathophysiology and patho- genesis of a disease almost invariably requires the devel- opment of an animal model. Concerning Symmers’ fibro- sis (clay pipestem fibrosis), different experimental hosts have been tried (Cheever et al. 2003), such as monkeys (Lichtemberg & Sadun 1968, Sadun et al. 1970, Lich- temberg et al. 1971, Damian et al. 1976, Farah et al. 2000), rabbits (Cheever et al. 1980), pigs (Hurst et al. 2000), and mice (Warren & DeWitt 1958, Andrade & Warren 1964, Cheever 1965, Warren 1968, Andrade 1987a, Andrade & Cheever 1993, Andrade et al. 1997). In this last animal model, during mild (one to two pairs of worms) and pro- longed (16 weeks or more) infections with Schistosoma mansoni, a picture mimicking “clay pipestem” fibrosis seen in humans with advanced schistosomiasis has been re- produced in outbred and in some strains of well-nour- ished inbred mice (Andrade & Cheever 1993), but not in undernourished outbred specimens (Coutinho et al. 1997). The importance of an adequate nutritional status for a healthy condition of the host is well recognized. Accord- ing to 1990 figures from the Disease Control Priorities Project in Developing Countries (Mason et al. 2003), it is estimated that 32% of the global burden of disease (mor- tality and morbidity) in these countries would be removed
Fig. 1: decreased parasite infectivity after opsonization with glycopro- tein-82 (gp82)-specific antibodies. Insect-derived metacyclic trypo- mastigotes (IMT) were incubated with control or anti-gp82 specific monoclonal antibodies and then either placed on the conjunctiva of anesthetized mice (A) or fed orally to BALB/c mice (B, C). DNA ex- tracted 11-13 days later from local draining tissues (parotid and sub- mandibular lymph nodes (LN) from conjunctivally challenged mice or gastric DNA from orally challenged animals) or distant sites (spleen) were used in Trypanosoma cruzi specific real-time polymerase chain reaction assays. In panel A, the numbers of T. cruzi molecular equiva- lents (ME) per 100 ng of DNA is significantly lower (asterisk means p < 0.04 by Mann-Whitney U test) in anti-gp82 opsonized MT-infected mice than in control opsonized MT-infected mice. Similarly, mice chal- lenged orally had markedly reduced amounts of T. cruzi DNA present in gastric samples after opsonization with anti-gp82 monoclonal an- tibody (MAb)-3F6 as compared to controls (B). In addition, systemic spread of parasites was inhibited by opsonization with anti-gp82, as seen in panel C where reduced parasite DNA was detectable in spleens recovered from mice that were infected with anti-gp82 MAb 3F6- opsonized MT compared with control opsonized MT (asterisk means p < 0.02 Mann-Whitney U Test). NC: negative control.
The catalytic activity of aromatase in Leydig cells was assayed by the formation of tritiated water from [1b- 3 H]-androstenedione as described previously . Briefly, TGF-b1-treated Leydig cells were incubated with 20 nM 1b- 3 H-androstenedione (New En- gland Nuclear Research Products, Boston, MA, USA) in serum- free medium for 6 h. Incubations were conducted in an identical fashion in the absence of cells to establish background values. Then the medium (600 m l) was extracted with 1,500 m l of ice-cold chloroform and centrifuged at 12,000 g at 4uC for 1 min. The aqueous phase was transferred to a vial containing 400 m l dextran- coated charcoal to remove the residual labelled and unlabelled steroids. The mixture was vortexed for 10 s and centrifuged at 15,000 g at 4 uC for 15 min. The supernatant was decanted, mixed with scintillation fluid and counted in a beta-spectrometer; thus, tritiated water formed during the aromatization of [1- 3 H]- androstenedione to estrogen was determined by measuring the radioactivity in the supernatant. Aromatase activity was expressed as rate of incorporation of tritium into water per mg protein per h for Leydig cells. Each experiment was conducted in triplicate and was repeated at least two times to ensure that the results were quantitatively reproducible.
Aberrant activation of fibroblasts to myofibroblasts is one of the hallmarks of renal fibrosisin chronic kidney diseases such as diabetes mellitus and hypertension. Activated myofibroblasts lead to accumulation of extracellular matrix, such as alpha-smooth muscle actin (α-SMA), fibronectin, and vimentin. This process is triggered by reactive oxygen species and inflamma- tory cytokines generated in injured kidney resident cells [1–4]. Transforming growth factor β (TGF-β) is recognized as a major pro-fibrotic cytokine of renal fibrosis. During renal fibrosis, TGF-β1 exerts its biological and pathological activities via Smad-dependent and Smad-inde- pendent signaling pathways. In canonical TGF-β/Smads pathway, the binding ofTGF-β1 to its receptor II (TβRII) activates the TGF-β receptor type I (TβRI) kinase. Then TβRI phosphory- lates Smad2 and Smad3 and subsequently, phosphorylated Smad2/Smad3 bind to Smad4 to form the Smad complex. This complex then translocates into the nucleus to regulate the fibrotic marker gene transcription, including type I collagen, α-SMA [5–7]. In non-canonical TGF-β/Smad pathway, TGF-β utilizes a multiple signaling pathway to regulate fibrotic gene expression through MAPKs pathway, Rho-like GTPase signaling pathways, phosphatidylinosi- tol-3-kinase/AKT-mTOR pathway, and Jak-Stat pathway [8–10].
nephrotoxicity: First, by its SOD mimetic effect , second, by preserving SOD activity  and third by directly scavenging peroxynitrite [34,35]. In all mechanisms, MnTBAP indirectly (SOD activity leads to decreased superoxide) or directly dimin- ished peroxynitrite levels . In contrast, FeTMPyP catalyzes the decomposition of peroxynitrite to nitrate in vivo [37,38,39]. In addition, it has been demonstrated the important role of iron in cisplatin-induced nephrotoxicity . We attempted here to quantify protein nitrotyrosine formation to confirm that MnTBAP and FeTMPyP was effective in its role as a peroxynitrite scavenger and we demonstrated that protein nitration level were significantly reduced from cisplatin treated group by both metalloporphyrins. Thus, our results supported the hypothesis that MnTBAP and FeTMPyP indeed reduced the level of peroxynitrite in cisplatin inducedin vivo model of kidney injury. Accumulating evidence indicates that peroxynitrite, formed from the diffusion-controlled reaction between nitric oxide and the superoxide radical represents a major oxidant and nitrating species triggering significant renal damage in this microenvironment . Perox- ynitrite is a powerful oxidant which is highly reactive towards biological molecules including protein and non-protein sulfhydryl, DNA, and membrane phospholipids [42,43,44]. Peroxynitrite is also stable enough to cross several cell diameters to reach targets before becoming protonated and decomposing .