GASTROENTEROLOGY

Neutrophil-Mediated Nitrosamine Role of Nitric Oxide in Rats

1992;103:1260-1266

Formation:

MATTHEW B. GRISHAM, KAREN WARE, HARRY E. GILLELAND, Jr., LINDA B. GILLELAND, COLETTE L. ABELL, and TAMAKI YAMADA Department of Physiology and Biophysics and Department University Medical Center, Shreveport, Louisiana

It is well known that chronic inflammation of the colon and rectum is associated with an increased risk of colorectal cancer, hut the mechanisms by which inflammation promotes neoplasia remain undefined. The authors propose that inflammatory neutrophils may produce carcinogenic nitrosamines via the L-arginine-dependent formation of nitrogen oxides such as nitric oxide. Therefore, the objectives of the study were to characterize the Larginine-dependent formation of nitrogen oxides by inflammatory (elicited) neutrophils using conditions that more closely mimic the extravascular (i.e., interstitial) compartment of the gut and to characterize the neutrophil-dependent N-nitrosation of a model amine to yield its nitrosamine derivative. In the absence of any metabolic activation, adherent, inflammatory neutrophils (2 X 10'cells) produced 12.8f 1.4pmol/L of nitrite during a 4hour incubation period. Omission of L-arginine and/or inhibition of nitric oxide synthase by the addition of 1 mmol/L NG-nitro-L-arginine methyl ester (L-NAME) resulted in 35%-78% inhibition of nitrite production, suggesting that nitrite was derived from nitric oxide. By comparison, neither circulating rat neutrophils nor elicited rat macrophages produced significant amounts of nitrite under the same conditions. Furthermore, elicited neutrophils (2 X 10’ cells) were capable of N-nitrosating 2&diaminonapthalene to yield its nitrosamine derivative l-naptho-2,34riazole (282 + 12 nmol/L) in a time- and cell-dependent pattern similar to that of nitrite production. Addition of a variety of antioxidants (e.g., ascorbic acid, reduced glutathione, a-tocopherol analog), 5-aminosalicylic acid, or L-NAME resulted in 80%-85% inhibition of neutrophil-mediated nitrosamine formation. Taken together, these data suggest that inflammatory neutrophils may represent an important metabolic source of endogenous carcinogens during times of active intestinal inflammation.

Microbiology

and Immunology,

Louisiana State

experimental data suggests that severe long-standing inflammation of the colon is associated with an increased risk of colorectal cancer.24 In addition, investigators have shown that inflammation enhances the formation of colonic tumors in experimental animals given known carcinogens.5’6 Despite these studies, the mechanisms by which inflammation promotes tumor formation remain poorly understood. It has been suggested that certain neutrophil-derived products may act as endogenous carcinogens or tumor promotors in viva.’ Recent studies have shown that neutrophils (and macrophages) are capable of generating the free radical nitric oxide (NO) via an L-arginine-dependent pathway.7-14 Nitric oxide is unstable in the presence of molecular oxygen and will rapidly and spontaneously decompose to yield a variety of nitrogen oxides such as nitrogen dioxide (NO,), dinitrogen trioxide (N,O,), and dinitrogen tetroxide (N,O,) as well as the stable anions nitrate and nitrite.15 It is well known that NO,, Nz03, and N,O, are potent nitrosating agents that will N-nitrosate primary and secondary amines to yield carcinogenic nitrosamines.15 Nitrosamines require metabolic activation to yield alkylating agents that have been shown to activate certain oncogenes via the covalent modification of certain DNA bases.” Although it is known that activated macrophages are capable of producing nitroso derivatives of certain amines in vitro, the ability of inflammatory (i.e., extravasated) neutrophils to synthesize these carcinogens has not been investigated.15 Therefore, the objectives of this study were to characterize the L-arginine-dependent formation of nitrogen oxides by inflammatory neutrophils using conditions that more closely mimic the extravascular (i.e., interstitial) compartment of the gut and to characterize the neutrophildependent A!-nitrosation of a model amine to yield its nitrosamine derivative. Materials and Methods Cell Preparations

A

ctive episodes of ulcerative colitis are characterized by infiltration of large numbers of neutrophils into the mucosal interstitium. This enhanced inflammatory infiltrate is accompanied by extensive injury to the mucosa.’ A growing body of clinical and

Male Sprague-Dawley for all experiments. Peritoneal 0 1992

bytheAmerican

rats (325-400 neutrophils

Gastroenterological 0016-5085/92/$3.00

g) were used were elicited Association

CHRONIC

October 1992

by the intraperitoneal injection of 20 mL of 1% (wt/vol) oyster glycogen in phosphate-buffered saline (PBS; pH 7.4). Cells,were harvested 16-18 hours later by peritoneal lavage using 100 mL of 37°C heparinized PBS. Cells were washed twice with cold PBS, and contaminating erythrocytes were removed by hypotonic lysis. Neutrophils were then washed three times in cold PBS, counted, and placed on ice. Cells were routinely found to be >60% neutrophils using this procedure. Circulating neutrophils were prepared from rat blood as previously described.” Briefly, erythrocytes were removed from heparinized blood by dextran sedimentation, and the leukocyte-rich supernatant was layered onto Histopaque 1077 (Sigma Chemical Co., St. Louis, MO; specific gravity, 1.077) and centrifuged at 400 X g for 40 minutes at 25°C. Contaminating erythrocytes in the neutrophil-rich pellet were removed by hypotonic lysis, and the neutrophils were washed three times with cold PBS, counted, and placed on ice. This preparation yielded cells that were >90% neutrophils. Peritoneal macrophages were elicited by the intraperitoneal injection of 15 mL of sterile thioglycollate medium (Difco Laboratories, Detroit, MI) prepared as described by the vendor. Cells were harvested 3 days later by peritoneal lavage using 100 mL of 37’C heparinized PBS. Cells were washed twice with PBS, suspended in room-temperature PBS, layered on Histopaque 1077, and centrifuged at 400 X g for 40 minutes at 25’C. Cells at the PBS-Histopaque interface were collected, washed twice with cold PBS, counted, suspended in PBS, and placed on ice. Cells prepared by this method were judged to be >85% macrophages/monocytes. Viability of all cell preparations after cell isolation and incubations was assessed using trypan blue exclusion as described by Salin and McCord.” Nitrite

Production

Varying numbers of neutrophils (elicited or circulating) and macrophages were suspended in a balancedsalt medium containing all electrolytes and salts present in Dulbecco’s modified Eagle medium (DMEM), 20 mmol/L HEPES (pH 7.4), 10% fetal bovine serum (FBS), 20 mmol/L glucose, 4 mmol/L glutamine, 1 mmol/L L-arginine, and 50 U/mL each of penicillin and streptomycin. For some experiments the NO-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 1 mmol/L) and/or metabolic activators such as phorbol myristate acetate (PMA), lipopolysaccharide (LPS), andN-formyl-methionyl-leucyl-phenylalanine (FMLP) were included in the reaction volumes. Cell suspensions (0.5 mL) were added to FBS-coated microtiter wells (1 cm’) and incubated for varying lengths of time at 37°C.” Coating tissue culture plastic with FBS has been shown to eliminate the nonspecific metabolic activation of neutrophils by the plastic and presents a surface more closely related to the interstitium.lg After the incubation period, the microtiter plates were centrifuged at 400 X g for 5 minutes, and the supernatants (0.5 mL) were removed and placed into glass tubes for nitrite determinations. Nitrite was quantified by adding 0.5 mL of the Griess reagent (0.1% naphthalene diamine dihydrochloride and 1% sulfanilamide in 5% H,POJ to each tube, incubating for 10 minutes at 25°C and measuring the absorbance at

INFLAMMATION

AND COLORECTAL

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543 nm.” The concentration of nitrite was calculated from a standard curve using sodium nitrite as the standard. For some experiments, phagocyte-mediated nitrate production was determined. Nitrate was quantified by first reducing nitrate to nitrite using bacterial nitrate reductase, then nitrite was measured using the Griess reagent as described above. The source of our nitrate reductase was Pseudomonas oleovorans (ATCC no. 8062; American Type Culture Collection, Rockville, MD).“’ Log-phase P. oleovorans was inoculated into 2-L flasks containing 500 mL of nutrient broth (Difco). After a 24-hour incubation at 25°C with continuous stirring, the bacteria were washed three times with PBS. The pellet from the final centrifugation was weighed and suspended in phenol red-free DMEM at a concentration of 10 mL/g wet wt. Aliquots (2 mL) were frozen at -7O’C. It has been determined that the nitrate reductase activity of these cells is stable under these storage conditions for more than 1 year.” On the day of the experiment, an aliquot of the frozen bacteria was thawed and placed on ice. Nitrate was quantitatively reduced to nitrite by incubating 0.5 mL of supernatants with 0.05 mL of bacterial suspension for 60 minutes at 37’C. After centrifugation at 10,000 X g for 5 minutes at 25°C supernatants were incubated with 0.5 mL of Griess reagent and processed as described above. Nitrate was calculated from a standard curve using sodium nitrate reduced by P. oleovorans as the standard. We found that the bacteria-associated enzyme is very sensitive to physical agitation and thus the bacterial suspension was not disturbed while on ice or during the 60 minutes of incubation at 37’C. Nitrosamine

Formation

Elicited neutrophils were suspended in the DMEM electrolyte solution containing 20 mmol/L HEPES (pH 7.4), 0.2 mmol/L 2,3_diaminonaphthalene (DAN), 20 mmol/L glucose, 4 mmol/L glutamine, 1 mmol/L L-arginine, and 50 U/mL each of penicillin and streptomycin. The cell suspension was then added to FBS-coated microtiter wells and incubated for 4 hours at 37’C. DAN is an amino compound that can be N-nitrosated to yield the highly florescent nitrosamine, l-naphtho(2,3)triazole(NAT).21 For some experiments the NO-synthase inhibitor L-NAME (1 mmol/L) and/or certain antioxidant enzymes and scavengers were included in the neutrophil suspension. After the incubation period, plates were centrifuged, and the supernatants were removed and placed into glass tubes. To the 0.5-mL supernatants, 2.5 mL of PBS was added, and the fluorescence was determined using an excitation wavelength of 375 nm and an emission wavelength of 450 nm.2* Concentrations of the nitrosamine derivative (NAT) were calculated from a standard curve using known concentrations of the purified triazole nitrosamine synthesized by the method of Wheeler et al.22 Statistical

Analysis

Standard statistical methods were used. All data are expressed as means + SEM. Statistical differences were identified using one-way analysis of variance, and multiple comparisons were performed using the least-significant-difference method or the Student’s t test for compari-

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GASTROENTEROLOGY

Vol. 103, No. 4

sons between two groups. Statistical significance was set at the 5% confidence level. Results Figure 1 shows that in the absence of metabolic activation, inflammatory (elicited) neutrophils produce a time- and cell-dependent increase in nitrite production such that 2 X lo6 cells produced 12.8 rt 1.43 pmol/L of nitrite during a 4-hour incubation period. In addition, we found that these cells release 9.14 f 2.6 pmol nitrate, which represents a ratio of nitrite to nitrate of 1.4 (data not shown). This ratio is similar to that reported for activated macrophages. l5 The viability of elicited rat neutrophils incubated for 4 hours at 37°C was 93% f 3% as judged by trypan blue exclusion.” Omission of L-arginine, inhibition of nitric oxide synthase by the addition of 1 mmol/L L-NAME, or a combination of the two resulted in 35%, 55% and 78% inhibition of nitrite formation, respectively (Figure 2). Figure 3 shows that addition of metabolic activators such as PMA or LPS enhanced nitrite production modestly (lo%-20%) but not significantly, whereas FMLP had no effect. By comparison, circulating neutrophils or elicited macrophages produced nitrite at levels of