Overview of the Lipid Peroxidation Measurements in Patients by the Enzyme-Linked Immunosorbent Assay Specific for the 4-Hydroxynonenal-Protein Adducts (4-HNE-ELISA).

Oxidative stress often affects the structure and metabolism of lipids, which in the case of polyunsaturated free fatty acids (PUFAs) leads to a self-catalysed chain reaction of lipid peroxidation (LPO). The LPO of PUFAs leads to the formation of various aldehydes, such as malondialdehyde, 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal, and 4-oxo-2-nonenal. Among the reactive aldehydes, 4-HNE is the major bioactive product of LPO, which has a high affinity for binding to proteins. This review briefly discusses the available information on the applicability of assessment options for 4-HNE and its protein adducts determined by immunosorbent assay (the 4-HNE-ELISA) in patients with various diseases known to be associated with oxidative stress, LPO, and 4-HNE. Despite the differences in the protocols applied and the antibodies used, all studies confirmed the usefulness of the 4-HNE-ELISA for research purposes. Since different protocols and the antibodies used could give different values when applied to the same samples, the 4-HNE-ELISA should be combined with other complementary analytical methods to allow comparisons between the values obtained in patients and in healthy individuals. Despite large variations, the studies reviewed in this paper have mostly shown significantly increased levels of 4-HNE-protein adducts in the samples obtained from patients when compared to healthy individuals. As with any other biomarker studied in patients, it is preferred to perform not only a single-time analysis but measurements at multiple time points to monitor the dynamics of the occurrence of oxidative stress and the systemic response to the disease causing it. This is especially important for acute diseases, as individual levels of 4-HNE-protein adducts in blood can fluctuate more than threefold within a few days depending on the state of health, as was shown for the COVID-19 patients.

Short Survey on the Protein Modifications in Plasma during SARS-CoV-2 Infection.

Although the COVID-19 pandemic has ended, it is important to understand the pathology of severe SARS-CoV-2 infection associated with respiratory failure and high mortality. The plasma proteome, including protein modification by lipid peroxidation products in COVID-19 survivors (COVID-19; n = 10) and deceased individuals (CovDeath; n = 10) was compared in samples collected upon admission to the hospital, when there was no difference in their status, with that of healthy individuals (Ctr; n = 10). The obtained results show that COVID-19 development strongly alters the expression of proteins involved in the regulation of exocytosis and platelet degranulation (top 20 altered proteins indicated by analysis of variance; p-value (False Discovery Rate) cutoff at 5%). These changes were most pronounced in the CovDeath group. In addition, the levels of 4-hydroxynonenal (4-HNE) adducts increased 2- and 3-fold, whereas malondialdehyde (MDA) adducts increased 7- and 2.5-fold, respectively, in COVID-19 and CovDeath groups. Kinases and proinflammatory proteins were particularly affected by these modifications. Protein adducts with 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) were increased 2.5-fold in COVID-19 patients, including modifications of proteins such as p53 and STAT3, whereas CovDeath showed a decrease of approximately 60% compared with Ctr. This study for the first time demonstrates the formation of lipid metabolism products-protein adducts in plasma from survived and deceased COVID-19 patients, significantly distinguishing them, which may be a predictor of the course of SARS-CoV-2 infection.

Editorial on Anticancer Antioxidants.

The current concepts of biomedicine consider oxidative stress to be one of crucial pathophysiological processes behind major stress- and age-associated diseases, including cancer [...].

Sensitivity of Osteosarcoma Cells to Concentration-Dependent Bioactivities of Lipid Peroxidation Product 4-Hydroxynonenal Depend on Their Level of Differentiation.

4-Hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological effects. Normal and malignant cells of the same origin express different sensitivity to HNE. We used human osteosarcoma cells (HOS) in different stages of differentiation in vitro, showing differences in mitosis, DNA synthesis, and alkaline phosphatase (ALP) staining. Differentiated HOS cells showed decreased proliferation (3H-thymidine incorporation), decreased viability (thiazolyl blue tetrazolium bromide-MTT), and increased apoptosis and necrosis (nuclear morphology by staining with 4',6-diamidino-2-phenylindole-DAPI). Differentiated HOS also had less expressed c-MYC, but the same amount of c-FOS (immunocytochemistry). When exposed to HNE, differentiated HOS produced more reactive oxygen species (ROS) in comparison with undifferentiated HOS. To clarify this, we measured HNE metabolism by an HPLC method, total glutathione (GSH), oxidized GSH (ox GSH), glutathione transferase activity (GST), proteasomal activity by enzymatic methods, HNE-protein adducts by genuine ELISA and fatty acid composition by GC-MS in these cell cultures. Differentiated HOS cells had less GSH, lower HNE metabolism, increased formation of HNE-protein adducts, and lower proteasomal activity, in comparison to undifferentiated counterpart cells, while GST and oxGSH were the same. Fatty acids analyzed by GC-MS showed that there is an increase in C20:3 in differentiated HOS while the amount of C20:4 remained the same. The results showed that the cellular machinery responsible for protection against toxicity of HNE was less efficient in differentiated HOS cells. Moreover, differentiated HOS cells contained more C20:3 fatty acid, which might make them more sensitive to free radical-initiated oxidative chain reactions and more vulnerable to the effects of reactive aldehydes such as HNE. We propose that HNE might act as natural promotor of decay of malignant (osteosarcoma) cells in case of their differentiation associated with alteration of the lipid metabolism.

The NRF2, Thioredoxin, and Glutathione System in Tumorigenesis and Anticancer Therapies.

Cancer remains an elusive, highly complex disease and a global burden. Constant change by acquired mutations and metabolic reprogramming contribute to the high inter- and intratumor heterogeneity of malignant cells, their selective growth advantage, and their resistance to anticancer therapies. In the modern era of integrative biomedicine, realizing that a personalized approach could benefit therapy treatments and patients' prognosis, we should focus on cancer-driving advantageous modifications. Namely, reactive oxygen species (ROS), known to act as regulators of cellular metabolism and growth, exhibit both negative and positive activities, as do antioxidants with potential anticancer effects. Such complexity of oxidative homeostasis is sometimes overseen in the case of studies evaluating the effects of potential anticancer antioxidants. While cancer cells often produce more ROS due to their increased growth-favoring demands, numerous conventional anticancer therapies exploit this feature to ensure selective cancer cell death triggered by excessive ROS levels, also causing serious side effects. The activation of the cellular NRF2 (nuclear factor erythroid 2 like 2) pathway and induction of cytoprotective genes accompanies an increase in ROS levels. A plethora of specific targets, including those involved in thioredoxin (TRX) and glutathione (GSH) systems, are activated by NRF2. In this paper, we briefly review preclinical research findings on the interrelated roles of the NRF2 pathway and TRX and GSH systems, with focus given to clinical findings and their relevance in carcinogenesis and anticancer treatments.

The Appearance of 4-Hydroxy-2-Nonenal (HNE) in Squamous Cell Carcinoma of the Oropharynx.

Tumor growth is associated with oxidative stress, which causes lipid peroxidation. The most intensively studied product of lipid peroxidation is 4-hydroxy-2-nonenal (HNE), which is considered as a "second messenger of free radicals" that binds to proteins and acts as a growth-regulating signaling factor. The incidence of squamous cell carcinoma of the oropharynx is associated with smoking, alcohol and infection of human papilloma virus (HPV), with increasing incidence world-wide. The aim of this retrospective study involving 102 patients was to determine the immunohistochemical appearance of HNE-protein adducts as a potential biomarker of lipid peroxidation in squamous cell carcinoma of the oropharynx. The HNE-protein adducts were detected in almost all tumor samples and in the surrounding non-tumorous tissue, while we found that HNE is differentially distributed in squamous cell carcinomas in dependence of clinical stage and histological grading of these tumors. Namely, the level of HNE-immunopositivity was increased in comparison to the normal oropharyngeal epithelium in well- and in moderately-differentiated squamous cell carcinoma, while it was decreasing in poorly differentiated carcinomas and in advanced stages of cancer. However, more malignant and advanced cancer was associated with the increase of HNE in surrounding, normal tissue. This study confirmed the onset of lipid peroxidation, generating HNE-protein adducts that can be used as a valuable bioactive marker of carcinogenesis in squamous cell carcinoma of the oropharynx, as well as indicating involvement of HNE in pathophysiological changes of the non-malignant tissue in the vicinity of cancer.

Cancer growth regulation by 4-hydroxynonenal.

While reactive oxygen species (ROS) gain their carcinogenic effects by DNA mutations, if generated in the vicinity of genome, lipid peroxidation products, notably 4-hydroxynonenal (HNE), have much more complex modes of activities. Namely, while ROS are short living and have short efficiency distance range (in nm or µm) HNE has strong binding affinity for proteins, thus forming relatively stable adducts. Hence, HNE can diffuse from the site or origin changing structure and function of respective proteins. Consequently HNE can influence proliferation, differentiation and apoptosis of cancer cells on one hand, while on the other it can affect genome functionality, too. Although HNE is considered to be important factor of carcinogenesis due to its ability to covalently bind to DNA, it might also be cytotoxic for cancer cells, as well as it can modulate their growth. In addition to direct cytotoxicity, HNE is also involved in activity mechanisms by which several cytostatic drugs and radiotherapy exhibit their anticancer effects. Complementary to that, the metabolic pathway for HNE detoxification through RLIP76, which is enhanced in cancer, may be a target for anti-cancer treatments. In addition, some cancer cells can undergo apoptosis or necrosis, if exposed to supraphysiological HNE levels in the cancer microenvironment, especially if challenged additionally by pro-oxidative cytostatics and/or inflammation. These findings could explain previously observed disappearance of HNE from invading cancer cells, which is associated with the increase of HNE in non-malignant cells close to invading cancer utilizing cardiolipin as the source of cancer-inhibiting HNE.

Adjuvant Cancer Biotherapy by Viscum Album Extract Isorel: Overview of Evidence Based Medicine Findings.

Within the integrative medicine one of the most frequently used adjuvant cancer biotherapies is based on aqueous mistletoe (Viscum album) extracts. Tumor growth inhibition, stimulation of host immune response and improvement of the quality of life are the positive effects of mistletoe therapy described in several preclinical and clinical studies. However, cumulative results of the evidence based medicine findings on such treatments are rarely given. Therefore, this paper evaluates the evidence based findings describing effects of the Viscum album extract Isorel in cancer therapy with respect to the type of therapy, stage and type of illness. This study presents cumulated data for 74 patients with different types and stages of cancer treated by Viscum album extract as adjuvant treatment to different conventional therapies, mostly combined surgery and radiotherapy. The biotherapy effectiveness was evaluated according to the outcome as (1) no major therapeutic improvement (15% of patients), (2) prevention of tumor recurrence (47% of patients) and (3) regression of cancer (38% of patients). Notably, there was no obvious health worsening during the follow up period at all. Thus, the results obtained for conventional anticancer therapies combined with adjuvant biotherapy based on Viscum album extract seem to be beneficial for the majority of cancer patients (85%) without serious side effects.

Assays for the measurement of lipid peroxidation.

Physical and emotional stress, metabolic alterations, carcinogenesis or inflammation are conditions that can trigger oxidative stress, which is defined as a balance shift of redox reactions towards oxidation, resulting in the increase of reactive oxygen species (ROS). ROS are continuously formed in small quantities during the normal metabolism of cell, however the overproduction of ROS is cytotoxic and damages macromolecules (DNA, proteins, sugars and lipids). Polyunsaturated fatty acids (PUFAs) that are esterified in membrane or storage lipids are subject to ROS-induced peroxidation resulting in the destruction of biomembranes. Final products of lipid peroxidation (LPO) are reactive aldehydes that are relatively stable and may diffuse far from the initial site of oxidative injury and act as second messengers or free radicals. The difference between physiological and pathological oxidative stress is often the occurrence of LPO and its final toxic products. In this chapter, two classes of methods for measurement of LPO are described. The first include assays for detection of LPO at the organismal level, while the second include molecular and cellular assays that reveal the mechanistic effects of LPO on the function, morphology and viability of the cells.

Superoxide dismutase and cytochrome P450 isoenzymes might be associated with higher risk of renal cell carcinoma in male patients.

Literature data support the hypothesis that oxidative stress and the accompanying antioxidant defense might play an important role in renal cell carcinoma (RCC) growth and progression. It is also known that the incidence of renal tumors is two times higher in men than in women. Thus, the aim of this study was to determine whether the oxidant/antioxidant profile of renal cell carcinoma tissue, adjacent to tumor tissue and nontumor tissue was different in male and female patients. Significantly higher lipid peroxidation (LPO) in renal cell carcinoma tissue compared to nontumor tissue was demonstrated only in male patients. Besides, gender-related difference in copper zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) in nontumor and renal cell carcinoma tissue was obtained at the level of transcription, translation and activity of these antioxidant isoenzymes. Morever, we demonstrated that the gene expression of 3 CYPs out of 7 was altered; CYP2D6 mRNA was decreased in both sexes while gender-related suppression of mRNA for CYP2E1 (women) and CYP2C19 (men) was observed. Taken together, these parameters might be potentially responsible for higher risk of renal cell carcinoma in men than in women.

Oxidative stress in small-for-gestational age (SGA) term newborns and their mothers.

This study used malondialdehyde (MDA) determination by HPLC and enzymatic assays for total serum peroxides and antioxidant capacity to evaluate oxidative stress in 47 healthy full-term small-for-gestational age (SGA) newborns vs 67 appropriate-for-gestational age (AGA) newborns. Blood samples were collected at delivery from umbilical cord artery and vein and from peripheral blood of the babies on the third day after birth. Blood samples of mothers were also collected and compared with blood of 29 normal non-pregnant women (NPW). Serum peroxide values were significantly higher in both groups of mothers than in NPW, decreasing towards the third day in AGA mothers, while persisting in SGA mothers. Antioxidant capacity of sera of both groups of mothers was lower than NPW. Both SGA mothers and babies had increased MDA at delivery, unlike AGA counterparts. MDA levels in umbilical vein were higher than in umbilical arteries, while immunohistochemistry revealed abundant presence of 4-hydroxynonenal (HNE)-protein adducts only in stroma of the SGA placenta. These results show that both mothers and babies are exposed to oxidative stress during and after delivery, which is more pronounced and persistent in the perinatal period of the SGA group, while lipid peroxidation in placenta could play a role in SGA pathophysiology.

The influence of 4-hydroxy-2-nonenal on proliferation, differentiation and apoptosis of human osteosarcoma cells.

The product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE) is known to cause cell death at high concentrations, while at lower concentrations it can influence cell proliferation and differentiation. In our experiments we used human osteosarcoma cells (HOS), to test the influence of HNE on cell proliferation, differentiation and induction of apoptosis. Apoptosis induction was estimated by TiterTACS TUNEL test. The cells were in parallel counted and the DAPI staining method was used to distinguish between apoptotic and necrotic cells as well as to define the proportion of cells in mitosis. To test the influence of HNE on HOS cell differentiation, cells were treated every second day with HNE. After 10 days, the cells were stained for alkaline phosphatase, a marker for osteoblast differentiation. Cell growth inhibition was caused by supraphysiological concentrations of 10 or 100 microM HNE, while apoptosis was induced with supraphysiological as well as by the physiological amount of the aldehyde (1 microM). Necrosis appeared when cells were treated with 10 or 100 microM, but not with 1 microM HNE. The proportion of cells in mitosis gradually declined with increased HNE concentration. Multiple exposures of HOS cells to 10 microM HNE prevented HOS cell differentiation. These results indicated that HNE inhibits proliferation and differentiation of HOS cells in the same concentration dependent manner as it causes apoptosis. We thus assume that HNE might be one of the important signaling molecules regulating the growth of the human osteosarcoma cells.

Oxidative burst and anticancer activities of rat neutrophils.

It is assumed that oxidative damage caused by reactive oxygen species (ROS) from activated neutrophil granulocytes may contribute to pathology of tumors. ROS are crucial in neutrophil-mediated tumor cell lysis. The present study is focused on the oxidative burst and antitumorous activities of neutrophils when challenged with Walker carcinoma W256. Survival and tumor growth dynamics were monitored in vivo, while tumor cell proliferation when mixed with neutrophils was studied in vitro together with the generation/release of neutrophil respiratory burst products, primarily 1O2. Neutrophils were collected upon Sephadex injection. The survival of Sephadex injected animals was slightly improved, while their tumors grew less than in controls. The presence of tumor cells in vitro activated neutrophils to produce singlet oxygen similar to phorbol ester. Neutrophils from Sephadex-bearing animals diminished tumor cell proliferation in vitro (measured by 3H-TdR incorporation), while neutrophils from Sephadex and the tumor-bearing animals did not show such activity in vitro. Our results confirm that in the case of rapidly growing tumors such as murine W256 carcinoma neutrophils have antitumorous effects in the early phase of tumor development.