Free radicals participate in both initiation and perpetuation of the inflammatory process observed in many diseases, trauma, and/or infection. Superoxide dismutase (SOD) is an enzyme that has anti-inflammatory capacity because of its ability to scavenge the superoxide free-radical, but its biological activity in rats and humans is poorly understood. SOD comprises a family of isoenzymes, which can be obtained from different sources with distinct anti-inflammatory efficacy. In addition, they seem to influence in a different way the various phases of the carrageenan-induced edema (CIE) model, in spite of having the same specific activity, metal at the active center, circulating half life, or molecular weight. Because the Cu,Zn-SOD from bovine erythrocytes (Be-SOD) was the first clinically used, and has been the most pharmacologically studied, we decided to compare its anti-inflammatory activity against a new Cu, Zn-SOD obtained from the marine yeast Debaryomyces hanseii (Dh-SOD). As reported elsewhere, the physicochemical and molecular characteristics of Dh-SOD indicate important similarities and differences with Be-SOD and other SOD sources, which may be reflected in different anti-inflammatory activity.

Several experimental animal models have been developed in order to study the anti-inflammatory activity of drugs. Among the most commonly used is the carrageenan-induced edema (CIE) model described originally by Winter et al. Based on this test, drugs can either affect the swelling to the early serotonin phase, or the late prostaglandin phase, or both. In addition, a very late amplification period is now being evaluated for the long-term effect of anti-inflammatory drugs. To our knowledge, no experimental work has been conducted to study the anti-inflammatory activity of SOD during the late amplification period, and has not been evaluated in animal experimental models once the lesion has been established (therapeutic mode).

Materials and Methods

SOD source

The enzyme Dh-SOD was obtained according to Reference 7. For this, the yeast biomass was produced in a sea-water formulated medium containing glucose 20 g/l, peptone 10 g/l, yeast extract 5 g/l and the pH adjusted to 5.0 with 0.1 N HCl. The culture was done in 60 l nalgene carboy sterilized with several washes of 70% ethanol (3 × 3 l), 1% NaHClO (1 l) for 30 min, sterile acid water (5% HCl) and finally rinsed with distilled water. A 7% chloride dioxide (HALOX E-100™, The Halox Co., Burlingame, CA, USA) solution was added (2 ml/l) to 30 l of culture medium to prevent the growth of contaminant and 10% of FG10 antifoam agent (Dow Corning) to prevent foaming. The inoculum was prepared in three 2-l flasks containing 500 ml of sterile culture medium by incubating at room temperature for 18 h at 100 rpm. The inoculum (1.5 l) was added to the fermentation bottle and the yeast allowed to grow under an air-flow of 3.3 l/min at room temperature during 48 h. After this incubation the cell biomass was removed by continuous centrifugation and the Dh-SOD enzyme obtained by disruption in a Bead-Beater as described previously. Be-SOD was obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Animals

The assays reported here were approved by the Animal Ethics Committee of CIBNOR. The female 130–170 g Wistar rats were purchased from a commercial supplier (Bioterio México, Mexico City). The animals were allowed to adapt for a 4-day period at the animal facility with water and food ad libitum. Only during the evaluation period that lasted 24 h were the animals restricted in food supply, but again water was provided ad libitum.

Induction of edema

Type IV carrageenan (1 type) was purchased from Sigma (St. Louis, MO, USA). A 1% solution in 0.9% saline was prepared 1 h before the experiment. One hundred milliliters of the carrageenan solution was administered intraplantary into the right hind paw of each animal (time 0).

Test groups

Ten groups of seven animals each were used to evaluate the preventive and therapeutic effect of Be-SOD and Dh-SOD. Their corresponding specific activity, by the nitroblue tetrazolium (NBT) method, was 4,400 U/mg and 8,328 U/mg, respectively. The enzymes were administered intraperitoneally in a 1 ml single dose 30 min before edema induction in the preventive study, and 90 min after carrageenan injection in the therapeutic evaluation.

Evaluation of edema

The volume of edema was evaluated using a mercury plethysmograph constructed by the Division of Technology of this Center. For this, the hind paw of the rat was marked with ink up to the tibiotarsalis joint and immersed into a mercury-filled cuvette which was connected to a transducer that conveyed an electrical signal to an ink recorder. The determinations of edema volume were performed at time 0 (carrageenan injection), 1, 2, 3, 4, 5, 6, 9, 12, and 24 h thereafter. Changes in edema volume of the hind paw are expressed in percentage of variation from time 0. For data analysis, the results of the serotonin phase are considered as the average of hours 1 and 2, while results of the prostaglandin phase as average of hours 3, 4, and 5; the amplification phase represents the average of hours 6 and 9.

Statistical analysis

Two-tail t test was used for comparison among groups in the therapeutic study protocol. Analysis of variance was used for the groups in the prophylactic protocol. The level of significance was 0.05%. All determinations were done by Quattro-Pro and STATISTICS computational programs.