Comparison of Activity of 8 Chemicals in Lipoxygenase Metabolism in Rat Liver and Lung

School of Public Health, China Medical University (Shenyang 110001) CAI Yuan Institute of Public Health, University of South Florida, APKulkarni, Metabolism of enzymatic activity such as the rate of formation of dioxin and glutamine such as peanut tannin nkLnetbookmark1 Abstract A comparative study of the activities of eight chemicals in the synergistic oxidation of lipoxygenase in rat liver and lung cell fluids was performed. The appropriate reaction conditions for liver enzymes are Tris buffered 2,3.5 mmol/L linoleic acid and 213 mg/L cytosol; the suitable reaction conditions for lung enzyme are Tris buffer, pH 6.5, 135 mmol/L linoleic acid and 223 mg/ L cytosol protein. Inhibitory nordihydroguaiaretic acid (NDGA) inhibited the response of hepatic and lupus oxysynthase oxidation of benzidine significantly and showed a dose-effect relationship. In addition to benzidine, tetramethylbenzidine, pyrogallol, guaiacol, tetramethyl phenylenediamine, nitridinobis(3-ethylbenzotriazole-6-sulfonic acid), o (b) 7 chemicals such as anisidine and p-phenylenediamine were also synergistically oxidized by liver and lung lipoxygenase. However, the liver enzyme activity only corresponds to 12% to 19% of the lung enzyme activity. The reason for this difference is that: (1) There are different lipoxygenase isoenzymes in liver and lung tissues: (2) Liver Lipoxygenase accounts for a low percentage of cytosolic protein and a small amount. The results suggest that liver and lung lipoxygenase may be an important way for the body to metabolize certain benzene ring structural chemicals.

Lipoxygenases are a group of isoenzymes that are iron-assisted and have the dual activity of dioxygenase and hydroperoxidase. Any substrate that has 1,4-cis-cis-pentadiene is inserted into one molecule of oxygen and converted to the corresponding hydroperoxide, which in turn is reduced to the hydroxy fatty acid. Usually the second step is slow. However, in the presence of a hydrogen donor, this step is significantly accelerated and the hydrogen donor is synergistically oxidized. Synergistic oxidation of hydrogen donors with lipoxygenase has been reported for styrene, benzidine, and other substances. In vitro experiments were performed to compare the activity of liver and lung lipoxygenase synergistic oxidation of eight exotic compounds. The results reported below.

1 Materials and Methods 1.1 Animals Female female rats weighing 200 to 260 g were provided at the Animal Room of the University of South Florida School of Medicine.

1.2 Reagents Linoleic acid (purity 99%) benzidine, pyrogallol, p-phenylenediamine, o-(anisidine), tetramethylbenzidine, guaiacol, tetramethyl diphenylenediamine, Nordihydroguaiaretic acid, both analytically pure, Sigma products. 2,2 Bis(3-ethylbenzotriazole-6-sulfonic acid) analytically pure, Aldrich products.

1.3 Preparation of subcellular components Rats were killed by ether anesthesia, and the liver and lungs were immediately perfused with ice-cold 9% NaCl solution to remove residual blood and dried. The liver and lungs were weighed and homogenized by adding pH 7.4, 100 mmol/L KCI, and 50 mmol/L phosphate buffer at a ratio of 50:100 (WVV). Centrifuge at 1000g 5min, 10000g and 100000g 1h differentially. Take 100,000 g centrifuged supernatant (cytosolic fraction) as a source of enzyme. The above operation is performed in an ice bath or under 4C conditions. Using the calf serum albumin as a standard, 15 mg/ml cytosolic proteins were quantified by the Lowry method.

1.4 Determination of suitable reaction conditions Set the experimental tube and control tube. The experimental tubes contained Tris buffer with different pH values, liver, or pulmonary cytosol proteins at concentrations of 1.0, 3.5, 60, and 8. 320 mg/L, and benzidine at 1 mmo/L, with a total volume of 3 ml in parallel tubes. The extracellular fluid protein is the same as the experimental tube. At the end of the test tube, the cytosolic fraction was added, and the optical density change due to the formation of the oxidation product benzidine diimine was immediately observed at a wavelength of 425 nm. Calculating the metabolic activity of the enzyme from 104/mol cm1 based on the extinction coefficient 6, the pH, linoleic acid concentration and cytosolic protein content at the fastest rate of benzidine oxidation were used as suitable reaction conditions, and other analyses were performed under this condition.

5 Determination of the synergistic oxidizing ability of liver and lung fluid enzymes by other chemical substances. Selection of chemical substances and calculation of enzyme activity. Reference M. Determination of the oxidation products of various chemical substances under the appropriate reaction conditions of liver and lung cytosolic enzymes The optical density at the maximum absorption wavelength was varied and the corresponding extinction coefficient was used to calculate the enzyme activity: tetramethyl 6 sulfonic acid) 412 mm, 3. chopping 104 mol/cm. The extinction coefficient of o-diethylanisole and p-phenylenediamine was not obtained. Therefore, the changes in optical density were observed at 470 nm and 485 nm, respectively, and the enzyme activity was determined accordingly.

16 Inhibitory effect of dehydroguaiaretic acid (NDGA) on enzyme activity NDGA was added to the experiment and control tubes at the same time as the other components at room temperature for 3 minutes. The hepatic or pulmonary cytosolic fractions were added to the experimental tubes. The reaction was started and the result of the determination 2 was as follows: 2.1 Effect of the reaction conditions on the oxidation rate of benzidine The pH value, linoleic acid concentration and cytosolic protein content all directly affected the oxidation rate of benzidine. The suitable conditions of liver enzymes were: pH 7.2, 3.5 mmol/L linoleic acid and 213 mg/L cytosol protein, the activity of catalyzed benzidine diimine was 3.75±014 nmol/Lmg-1 protein; lung enzyme catalysis The suitable conditions for the same reaction are: pH 6. In addition, the activities of both enzymes are reduced to varying degrees.

2.2 Effect of NDGA on oxidation rate of benzidine (Table 1) As can be seen from Table 1, NDGA has a significant inhibitory effect on the formation of benzidine diimide, an oxidation product of benzidine, and shows a dose-effect relationship. This reflects the decrease in lipoxygenase activity in rat liver and lung fluid under the action of NDGA. In comparison, liver enzymes are more sensitive to NDGA.

Table 1 Inhibitory effect of NDGA on oxidation product benzidine diimine formation (X±S) Liver enzyme lung enzyme Note: Activity comparison of liver and lung lipoxygenase synergistic oxidation chemicals in nmol/Lg I protein 23 rat (Table 2) Table 2 Liver and lung cytosolic lipoxygenase metabolism in rat Liver 8 Activity comparison (X Shi S) Chemicals Liver enzymes Lung enzymes Liver enzyme activity/Pneumoenzyme activity Beniline tetramethylbenzidine Phyrogallol guaiacol tetramethyl phenylenediamine nitidine bis (ethyl benzotriazole sulfonic acid) o (2) anisidine p-phenylenediamine 1 protein 3 accounts for a smaller proportion.

Lipoxygenases are present in plants and in a variety of mammalian tissues, including humans, and have at least three isoforms of 5-lipoxygenase, 12-lipoxygenase, and 15-lipoxygenase [3]. Kulkarni et al. reported that soybean lipoxygenase can synergistically oxidize benzidine and other chemical substances b4]. The results of this experiment show that the rat liver and lung cell fluid components can oxidize benzidine to benzidine diimine, and the reaction rate is closely related to pH value, linoleic acid concentration, cytosolic protein content, and is classically affected by enzymes. Inhibition of the inhibitor NDGA demonstrated that lipoxygenase catalyzes this reaction.

The ability of liver enzymes to synergistically oxidize 8 chemicals such as benzidine is significantly lower than that of lung enzymes, and the highest is less than 20% of lung enzymes. The reasons may be: (1) There are different isoenzymes in liver and lung. Yamamoto summarized the distribution of lipoxygenase isoenzymes in different tissues of pigs. In the lung, 5-lipoxygenase is predominant, and 12-lipoxygenase activity is weak; in the liver, only 12-lipoxygenase [5] is seen from this result. The suitable pH values ​​for liquefaction-catalyzed reactions were 7.2 and 65, respectively, which were not the same. Therefore, it is speculated that the distribution of lipoxygenase isozymes in liver and lung of rats is similar to that of pigs. (2) The ratio of lipoxygenase in the liver and lungs is different in the cytosol protein. The data show that the activity of porcine liver enzyme is only about 27% of the activity of the lung enzyme. Under the suitable reaction conditions of this experiment, the amount of protein in liver and lunar cells as the source of enzyme is 213mg/L but the direct substrate is oil. The requirement of the acid was 86 times that the liver enzyme's catalytic activity was much lower than that of the lung enzyme. Enzyme inhibitor NDGA inhibited liver enzyme activity by 100% at 5 mol/L, but did not completely inhibit lung enzyme activity at 25 mol/L (92.4%) suggesting that the amount of liver enzymes is lower than that of lung enzymes, ie, in liver cytosol The previous data combined with the protein found that different sources of lipoxygenase have the ability to synergistically oxidize benzene ring structural chemicals. Therefore, the enzyme may be closely related to the outcome of such substances in the body and the biological activity. Due to the presence of carcinogens such as benzidine, the toxicological significance of this metabolic pathway deserves further investigation.