Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and leads to the generation of reactive oxygen species (ROS). Heme oxygenase (HO, EC 1.14.99.3) plays a protective role against oxidative stress in mammals, but little is known about this issue in plants. Here, we report for the first time the response of HO in leaves of soybean (Glycine max L.) plants subjected to UV-B radiation. Under 7.5 and 15 kJ m(-2 )UV-B doses, HO, catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) activities were increased and the production of thiobarbituric acid reactive substances (TBARS) regain control values after 4 h of plant recuperation. Treatment with 30 kJ m(-2) UV-B provoked a decrease in these antioxidant enzyme activities. Immunoblot analysis showed a 4.3 and 3.7-fold increase in HO-1 protein expression after irradiation with 7.5 and 15 kJ m(-2), respectively. HO-1 transcript levels were enhanced (up to 77%) at these doses, as assessed by semi-quantitative RT-PCR. These data demonstrated that increased HO activity was associated with augmented protein expression and transcript levels. Plants pre-treated with the antioxidant ascorbic acid did not show the UV-B-induced up-regulation of HO-1 mRNA, but hydrogen peroxide treatment could mimic this reaction. Our results indicate that HO is up-regulated in a dose-depending manner as a mechanism of cell protection against oxidative damage and that such response occurred as a consequence of HO-1 mRNA enhancement involving ROS.

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Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.

Abstract.We isolated and characterized cell lines resistant to aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) derived from a murine adenocarcinoma and studied cross resistance with other injuries. The most resistant clones were numbers 4 and 8, which exhibited 6.7-and 4.2-fold increase in resistance respectively. Several characteristics were altered in these clones. A 2-fold increase in cell volume, higher cell spreading, and a more fibroblastic, dendritic pattern, were the morphology features that led us to think they could have different adhesive, invasive or metastatic phenotypes. The amount of porphyrins synthesized per cell in the resistant clones was similar to the parental line but, when it was expressed per mg protein, there was a 2-fold decrease, with a higher proportion of hydrophilic porphyrins. These cells were not cross-resistant to photosensitization with Benzoporphyrin derivative and Merocyanine 540, but exhibited a slight resistance to exogenous protoporphyrin IX treatment. Both clones displayed higher protein content and increased number of mitochondria, together with a higher oxygen consumption. The distinctive features found in the resistant lines led as to think how to exploit the changes induced by PDT treatment to target surviving cells. Those hypoxic cells can be also a preferential target of bioreductive drugs and hypoxia-directed gene therapy, and would be sensitive to treatment with other photosensitizers.

The short- and long-term pro-oxidant effect of protoporphyrin IX (PROTO) administration to mice was studied in liver. A peak of liver porphyrin accumulation was found 2 h after the injection of PROTO (3.5 mg/kg, i.p.); then the amount of porphyrins diminished due to biliar excretion. After several doses of PROTO (1 dose every 24 h up to 5 doses) a sustained enhancement of liver porphyrins was observed. The activity of delta-aminolevulinic acid synthetase was induced 70-90% over the control values 4 h after the first injection of PROTO and stayed at these high levels throughout the period of the assay. Administration of PROTO induced rapid liver damage, involving lipid peroxidation. Hepatic GSH content was increased 2h after the first injection of PROTO, but then decreased below the control values which were maintained after several doses of porphyrin. After a single dose of PROTO, Cu-Zn superoxide dismutase (SOD) was rapidly induced, suggesting that superoxide radicals had been generated. Increased levels of hydrogen peroxide coming from the reaction catalyzed by SOD and lipid peroxides as a consequence of membrane peroxidation, induced the activity of catalase and glutathione peroxidase (GPx), while decreased GSH levels induced glutathione reductase (GRed) activity. However after 5 doses of PROTO, the activity of SOD was reduced reaching control values. GPx and catalase activities slowly went down, while GRed continued increasing as long as the levels of GSH were kept very low. TBARS values, although lower than those observed after a single dose of PROTO, remained above control values; Glutathione S-transferase activity was instead greatly diminished, indicating sustained liver damage. Our findings would indicate that accumulation of PROTO in liver induces oxidative stress, leading to rapid increase in the activity of the antioxidant enzymes to avoid or revert liver damage. However, constant accumulation of porphyrins provokes a liver damage so severe that the antioxidant system is compromised.