Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F 2 -isoprostanes are widely used as markers of oxidative stress in vitro and in vivo . This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coeffi cients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

Acrolein is a toxic unsaturated aldehyde and widespread environmental pollutant produced during lipid peroxidation and also by burning of tobacco or liquid fuels. Inhalation or dermal exposure to acrolein could be toxic to organisms. This very reactive aldehyde has a strong affinity for binding to proteins thus forming pathogenic protein-adducts. In the present study we have analyzed formation of bioreactive acrolein-protein adducts in bovine serum albumin solution exposed to exhaust gases of mineral diesel fuel and of mineral diesel fuel supplemented with different amounts of a novel diesel fuel additive denoted Ecodiesel (produced by a genuine procedure of recycling of plant oils used for food preparation). The effects of acrolein-protein adducts were tested on human microvascular endothelial cells and on human osteosarcoma cells that are sensitive to bioactivities of lipid peroxidation products. The results have shown a reduction of the bioreactive acrolein in exhaust gases when mineral diesel was supplemented with 5-20% Ecodiesel. Moreover, acrolein-protein adducts obtained from mineral diesel supplemented with Ecodiesel were less toxic than those obtained from mineral diesel alone. Thus, we assume that supplementing mineral diesel fuel with Ecodiesel would be of benefit for the use of renewable energy, for environment and for human health due to reduced environmental pollution with bioreactive acrolein.

Oxidative stress, i.e., excessive production of oxygen free radicals and reactive oxygen species, leads to lipid peroxidation and to formation of reactive aldehydes which act as second messengers of free radicals. It has previously been shown that oxidative stress may be involved in the transcriptional regulation of cytomegalovirus (CMV) immediate early promoter, involved in viral reactivation from latency. In the current study we used a plasmid containing the yellow fluorescent protein (YFP) gene under the control of CMV-1 promoter to monitor the influence of hydrogen peroxide and reactive aldehydes, 4-hydroxy-2-nonenal (HNE) and acrolein, on CMV-1 promoter activation in human embryonic kidney cells (HEK293). While acrolein was ineffective, hydrogen peroxide slightly (50 %) stimulated the CMV promoter. In contrast, HNE had a strong, up to 3-fold, enhancing effect on the CMV-1 promoter within four as well as after 24h of treatment. The most effective was the treatment with 24 microM HNE. This effect of HNE suggests that stressful conditions associated with lipid peroxidation could lead to CMV activation.

Bone regeneration is a process of vital importance since fractures of long bones and large joints have a highly deleterious impact on both, individuals and society. Numerous attempts have been undertaken to alleviate this severe medical and social problem by development of novel bioactive materials, among which bioactive glass is the most attractive because of its osteoconductive and osteostimulative properties. Since lipid peroxidation is an important component of systematic stress response in patients with traumatic brain injuries and bone fractures, studies have been undertaken of the molecular mechanisms of the involvement of 4-hydroxynonenal (HNE), an end product of lipid peroxidation, in cellular growth regulation. We found that HNE generated in bone cells grown in vitro on the surfaces of bioactive glasses 45S5 and 13-93. This raises an interesting possibility of combined action of HNE and ionic bioglass dissolution products in enhanced osteogenesis probably through a mitogen-activated protein kinase (MAPK) pathway. While the proposed mechanism still has to be elucidated, the finding of HNE generation on bioglass offers a new interpretation of the osteoinducting mechanisms of bioglass and suggests the possibility of tissue engineering based on manipulations of oxidative homeostasis.

Breast cancer is a leading cause of mortality and morbidity in women, mostly due to high metastatic capacity of mammary carcinoma cells. It has been revealed recently that metastases of breast cancer comprise a fraction of specific stem-like cells, denoted as cancer stem cells (CSCs). Breast CSCs, expressing specific surface markers CD44(+)CD24(-/low)ESA(+) usually disseminate in the bone marrow, being able to spread further and cause late metastases. The fundamental factor influencing the growth of CSCs is the microenvironment, especially the interaction of CSCs with extracellular matrix (ECM). The structure and function of ECM proteins, such as the dominating ECM protein collagen, is influenced not only by cancer cells but also by various cancer treatments. Since surgery, radio and chemotherapy are associated with oxidative stress we analyzed the growth of breast cancer CD44(+)CD24(-/low)ESA(+) cell line SUM159 cultured on collagen matrix in vitro, using either native collagen or the one modified by hydroxyl radical. While native collagen supported the growth of CSCs, oxidatively modified one was not supportive. The SUM159 cell cultures were further exposed to a supraphysiological (35 microM) dose of the major bioactive lipid peroxidation product 4-hydroxynonenal (HNE), a well known as 'second messenger of free radicals', which has a strong affinity to bind to proteins and acts as a cytotoxic or as growth regulating signaling molecule. Native collagen, but not oxidised, abolished cytotoxicity of HNE, while oxidized collagen did not reduce cytotoxicity of HNE at all. These preliminary findings indicate that beside direct cytotoxic effects of anticancer therapies consequential oxidative stress and lipid peroxidation modify the microenvironment of CSCs influencing oxidative homeostasis that could additionally act against cancer.