POSSIBLE FUNCTIONAL IMMUNOTOXICITY OF ACRYLONITRILE (VCN)

Pharmacological Research, Vol. 37, No. 2, 1998

POSSIBLE FUNCTIONAL IMMUNOTOXICITY OF ACRYLONITRILE ( VCN ) F. M. HAMADAU , A. H. ABDEL-AZIZ, A. R. ABD-ALLAH and A. E. AHMED† Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar Uni¨ ersity, Nasr-City, Cairo, Egypt and †Department of Pathology, UTMB, Gal¨ eston, TX, USA Accepted 6 No¨ ember 1997

Acrylonitrile Žvinyl cyanide, VCN., an environmental pollutant, has been shown to be an animal and human carcinogen particularly for the GIT. In a previous work done in our laboratory, VCN induced immunosuppressive effects as indicated by a decrease in plaque forming cell ŽPFC. response to SRBCs Žsheep red blood cell. immunization, a marked depletion of spleen lymphocyte subsets by flow cytometric analysis as well as bacterial translocation of the normal flora leading to brachial lymph node abscess. This work was carried out to evaluate the systemic andror local immunotoxic potential of VCN. Acrylonitrile Ž2.7 mg kgy1 dayy1 . was given to CD-1 mice once daily for 5, 10 and 15 days. Immunohistochemical assessment of the number of cells capable of producing IgA in different intestinal compartments Žduodenum, jejunum and ileum. revealed a significant decrease following VCN treatment. On the contrary, Bromodeoxyuridine ŽBrdU. incorporation in gut epithelial cells Žduodenum and ileum. showed a significant increase in the same VCN-treated groups of animals. On the other hand, w 3 Hxthymidine uptake was significantly decreased in splenocytes stimulated with phytohemaglutinin ŽPHA., Concanavalin-A ŽCon-A. and Lipopolysaccharide ŽLPS. and derived from animals treated with VCN. The effects of VCN were started after 5 days and increased up to 15 days of daily treatment in most of the investigated parameters. The results suggested that VCN has a profound immunosuppressive effect either systemically or locally which could be a contributing factor in its GIT carcinogenicity. Q 1998 The Italian Pharmacological Society

INTRODUCTION Acrylonitrile ŽCH 2 sCH]CN, vinyl cyanide, VCN. is a monomer used in many industries including plastics, plastizers, rubber, acrylic and modacrylic fibers w1x. An important use of VCN in clinical practice is in the manufacture of dialysis tubings w2x. It is also used in the synthesis of artificial membranes for pancreatic islets incapsulation w3x. VCN is detected in water, air and foods contained in cans fabricated from acrylonitrile copolymers w4x. It is also found in car exhaust and cigarette smoke w5x. VCN is demonstrated to be an animal carcinogen w6x, mutagen w7x and teratogen w8x. Different studies demonstrated that VCN induces mucosal necrosis and ulcerogenic damage to the stomach and intestine w9, 10x. VCN was also shown to directly interact U

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with intestinal DNA and enhance its modifications w11x. The gastrointestinal tract was considered as a major target organ for VCN-tumorgenicity in both experimental animals and humans w12]14x. Ahmed et al. w15x reported a great humoral and cell-mediated immunosuppresive potential of VCN in CD-1 mice. Acrylonitrile markedly decreased the percentage of lymphocytes and the lymphocyte subsets ŽT and B cells. in the spleen cell suspension. Also, the Th and Ts lymphocytes were obviously inhibited. VCN decreased the number of plaque forming cells ŽPFC. in the splenocytes of the treated mice. Moreover, brachial lymph node abscess, lung abscess and severe enlargement in Peyer’s patches in the small intestine were observed. Therefore, this study was carried out to investigate the possible functional immunotoxicity of VCN systemically by assessing the lymphoproliferative capability Žas indicated by w 3 Hxthymidine uptake. of the splenocytes of treated mice in response to difQ1998 The Italian Pharmacological Society

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ferent mitogens; Concanavalin-A ŽCon-A., Phytohemaglutinin ŽPHA. and Lipopolysaccharide ŽLPS. as well as locally by evaluating the number of IgA-producing cells in the different compartments of the gastrointestinal tract. The rate of the gut epithelial cells proliferation Žindicated by Bromodeoxyuridine, BrdU, incorporation. was assessed immunohistochemically. MATERIALS AND METHODS

Chemicals VCN Ž99%, ds 0.806., was inhibited with 35 ppm of quinone monomethyl ether, purchased from Aldrich Chemical Co., Inc, Milwaukee, WI, USA. BrdU, Con-A, LPS and PHA were obtained from Sigma Chemical Co., St. Louis, MO, USA. w 3 HxThymidine Žspecific activity 331 mCi mgy1 . was obtained from Amersham Life Science, Buckinghamshire, England. Liquid scintillation cocktail was purchased from Clear-Count, True-Lab., Liberty Ville, IL, USA. All the other used chemicals were of commercially available analytical grade.

Animals Male CD-1 mice Ž30]35 g B.W.. were obtained from Charles River laboratory ŽWilmington, MA, USA. and housed in stainless steel wire bottom cages. They were given Purina certified rodent chow ŽPurina, MA, USA. and water ad libitum and maintained in an air-conditioned room on a 12-h lightrdark cycle at 22 " 28C. The mice were acclimatized in our animal facility for about 1 week before experimentation.

Animal treatment Dosing solutions of VCN were freshly prepared in distilled water and given p.o., once daily at a dose of 2.7 mg kgy1 to three groups of mice for 5, 10 and 15 consecutive days, respectively. The dose of VCN was 10% of its LD50 w12x. The control group was given distilled water concurrently with the treated groups for 10 days. All mice were injected i.p. with BrdU Ž100 mg kgy1 ., 1 h before killing the animals. Each group of mice was killed by cervical dislocation at each specific time interval post-treatment and the mice were immediately immersed in 70% ethanol. In a biological safety cabinet, the spleens were aseptically dissected out and placed in sterile Hank’s Balanced Salt solution ŽHBSS. Žeach spleen was processed separately.. Spleen cell suspensions were prepared according to Deyo and Kerkvliet w16x and subjected to the mitogen response assay. On the other hand, a small part Ž2 cm length. of each intestinal compartment Žduodenum, jejunum and ileum., from each mouse, was also cut off and fixed for immunohistochemical counting of IgA-producing cells and assessment of BrdU incorporation in gut epithelial cells.

Methods Mitogen response assay. This assay depends on the mitogenic stimulation of lymphocyte proliferation as measured by tritiated thymidine, w 3 Hxthymidine, uptake w17x. Spleen cell suspension was adjusted to 2 = 10 6 viable cells per ml using the trypan blue exclusion technique. Then, 0.1 ml Ž2 = 10 5 cells. from each cell per ml of suspension was placed in a 96-well polystyrene culture plate. PHA, Con-A and LPS were added in a volume of 50 m l in concentrations of 2 m g per well, 2 m g per well and 200 ng per well, respectively. The final volume of all the wells was adjusted to 0.2 ml with RPMI1640. The plates were incubated in 5% CO 2 incubator at 378C for 72 h. Eighteen hours before cell harvesting Žthe end of the incubation period., w 3 Hxthymidine was added in a 20-m l volume Ž1.0 m Cirwell.. The cells were harvested using cell harvester, on glass fiber filter strips Ž240-1 Cambridge Technology, Inc... The glass filter strips were collected in scintillation vials which were kept overnight to dry at room temperature. Then 5 ml of scintillation cocktail was added to each vial. Incorporated w 3 Hxthymidine was counted in a bCounter for 2 min. The arithmetic mean of CPM Žaverage count per minute. corresponding to each mitogen replicates and its standard error were calculated. Immunohistochemical counting of IgA-producing cells in mouse small intestine [18]. The intestinal tissues were immediately placed in vials containing 4% paraformaldehyde in phosphate buffer saline ŽPBS. and fixed overnight with gentle shaking. The tissues were transfered to other vials containing 25% sucrose in PBS and kept overnight with gentle shaking as well. Again the tissues were transfered to appropriate cassettes and placed in 10% ethanol until processed to sectioning. Tissue sections were hung on a slide holder and immersed in three coplin jars sequentially containing xylene for 10 min, absolute ethanol for 2 min and 95% ethanol for 2 min. The slides were gently washed with water then placed in a coplin jar to which 50 ml of 3% H 2 O 2 was added and kept for 30 min. The slides were washed immediately and placed on a horizontal slide holder. Ten percent normal goat serum ŽNGS., in phosphate buffered saline was added dropwise to cover the whole section. NGS was then decanted and the sections were wiped around to decrease staining background. Monoclonal antibodies ŽAnti-IgA. conjugated with horseradish peroxidase was diluted with PBS ŽpH 7.4. to 1:40 and two drops were added on top of the tissue sections and placed in a humidifier for 90 min. The slides were washed with PBS and the mouse complex ŽMCX. Žantibody to counteract the non-specific binding. was diluted Ž1:100. with PBS and added Žtwo drops. on top of the tissue sections and placed in a humidifier for 60 min. Again the slides were washed with PBS and two drops of 3,39-diaminobenzidine tetrahydrochloride

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ŽDAB. were added on top of the sections for 2 min. DAB was decanted and the sections were washed with deionized water for 5 min. All the sections were counter stained with Mayer’s hematoxylin for 4 min. The stained sections were dehydrated, placed in a coplin jar containing xylene, wiped around and covered with glass coverslips. Stained sections were examined under light microscope for scoring the positively stained cells Žbrown stained. in 40 villi of each intestinal section Žchosen randomly from different parts of the sections. w18x. The results were expressed as mean " SEM of the positively stained cells per 40 villi. Immunohistochemical assessment of bromodeoxyuridine (BrdU) incorporation in mice gut epithelial cells [20]. Deparaffinized sections were placed in a coplin jar containing 3% H 2 O 2 and kept at room temperature for 30 min. Slides were washed with deionized water, imbedded in 0.2 N HCl and kept for 30 min at room temperature. The slides were washed again with deionized water and to which proteinase K 7% in PBS Ž50 ml. was added and incubated at 378C for 15 min in a water bath. All the slides were rinsed with PBS. Then 10% NGS was added, decanted and the sections were wiped around as previously mentioned in counting IgA-producing cells. Primary monoclonal anti-BrdU antibodies were added Žwithout dilution. on top of the sections and kept in the humidifier for 90 min. The slides were washed with PBS for 2 min and goat antimouse secondary antibodies conjugated with horseradish peroxidase was diluted with PBS to 1:50 and added Žtwo drops. on top of the sections then kept in the humidifier for 60 min. The MCX was diluted, added and the slides were washed as described previously. DAB was added, decanted and the sections were washed with deionized water. All the slides were counter stained by Mayer’s hematoxillin as exactly mentioned in immunohistochemical counting of IgA-producing cells. Stained sections were examined

under light microscope for scoring the positively stained nuclei Žbrown stained. in 40 villi randomly chosen from different parts of each section in the small intestinal compartments Žduodenum, jejunum and ileum. w19, 20x.

Statistical analysis The GRAPH PAD ŽISI Software, Philadelphia, PA, USA. computer program was used. Data were expressed as means " SEM. The Student’s t-test was used for statistical analysis between groups. The 0.05 level of probability was used as the criterion for significance.

RESULTS

Mitogen response in mice splenocytes

Mice treated with VCN Ž2.7 mg kgy1 dayy1 . for 5, 10 and 15 days showed a significant decrease in w 3 Hxthymidine uptake by their splenocytes after stimulation with Con-A Ž68.1%, 67.8% and 78.6%, respectively. and LPS Ž34.5%, 60.3% and 57.4%, respectively.. On the other hand, the uptake of w 3 Hxthymidine by the splenocytes after stimulation with PHA was markedly reduced only after 15 days of VCN treatment by 32.5% of the control value ŽTable I..

IgA-producing cells in small intestinal compartments The counts of IgA-producing cells in the duodenum of mice treated with VCN Ž2.7 mg kgy1 dayy1 . for 5, 10 and 15 days were significantly decreased by 56%, 72.8% and 77.3%, respectively, from the control value wFig. 1Ža.x. Also, the counts of IgA-producing cells were markedly decreased in the jejunal compartment of mice treated with VCN for 5, 10 and 15 days by 44.4%, 57.3% and 66.9%, respectively wFig. 1Žb.x. In addition, the same treatment

Table I Time course study for the effect of VCN administration on [ 3 H]-thymidine uptake of mice splenocytes under the influence of different mitogens in ©itro Mitogens

PHA Con-A LPS

Days of VCN treatment. [Counts per min (CPM)]

Control 1331 " 163 14 927 " 972 1225 " 112

5

10

15

1203 " 265 4756U " 532 802.7U " 87

1434 " 35 4792U " 946 486U " 1.5

898U " 32 3197.6U " 448 522U " 30.8

VCN was given orally once daily Ž2.7 mg kgy1 dayy1 . for the specified time intervals and mice were killed 24 h after the last dose. VCN-treated mice Ž n s 4., while the control group consisted of eight mice killed 10 days after starting the experiment. Splenocytes derived from VCN-treated mice as well as controls were cultured in 96-well culture plates, then the mitogens, e.g. Con-A, PHA and LPS were added in concentrations of 2 m g per well, 2 m g per well and 200 ng per well, respectively. The cultures were then incubated in a 5% CO 2 incubator at 378C for 72 h. w 3 Hxthymidine was added to the cultures 18 h before cell harvesting at a concentration of 1 m Ci per well. Data are presented as mean ŽCPM. " SEM Žthe mean of triplicate cultures ŽCPM. for each animal was calculated from which the mean ŽCPM. " SEM for each group of animals was calculated .. U Significantly different from control group at P- 0.05.

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Fig. 1. Time course study for the effect of VCN administration on lgA producing cells in mice duodenum Ža., jejunum Žb. and ileum Žc.. VCN was given orally once daily in a dose of 2.7 mg kgy1 for 5, 10 and 15 days. The animals were killed 24 h after the last dose. VCN-treated mice s 4rgroup. Control s eight mice were killed after 10 days of starting the experiment. Data are presented as percentage of control "SE Žthe number of positively stained cells per 40 villi.. U Significantly different from control at P - 0.05.

inhibited significantly the number of IgA-producing cells in the ileum of mice treated for 10 and 15 consecutive days amounting to 61.5 and 59.9%, respectively wFig. 1Žc.x.

BrdU incorporation by mice intestinal epithelial cells The number of epithelial cells capable of incorporating BrdU was significantly increased in the duodenal compartment of mice treated with VCN Ž2.7 mg kgy1 dayy1 . for 5, 10 and 15 days amounting to 300%, 328.8% and 342.6%, respectively of the control value ŽTable II.. In the jejunal compartment of the same groups of animals, the number of cells incorporating BrdU, showed no significant change from that of control mice. Furthermore, BrdU incorporations by epithelial cells in ileal compartments were markedly increased by about a 1.6 and 1.7-fold increase of control in mice treated with VCN Ž2.7 mg kgy1 dayy1 . for 10 and 15 days, respectively ŽTable II..

DISCUSSION Recently, it was demonstrated by Ahmed et al. w15x the possible immunosuppressive effect of VCN on both the humoral and cell-mediated immunity in CD-1 mice. The authors reported that VCN significantly decreased the percentage of lymphocytes in spleen cell suspension as well as the lymphocyte subsets ŽT and B cells.. Also, Th and Ts lymphocytes were markedly decreased. The number of PFC, as a response to sheep red blood cells immunization, was decreased in VCN-treated mice. Moreover, they reported the presence of brachial lymph node abscess, lung abscess and severe enlargement in Payer’s patches in the small intestine of VCN-treated mice. On the other hand, many studies demonstrated that GIT is a target organ of VCN toxicity andrtumorgenicity w9, 10, 21x. Therefore, the present work was designed to clarify the possible functional immunotoxicity of VCN on the systemic immune function of

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Table II Time course study for the effect of VCN administration on the rate of BrdU incorporation in epithelial cells of different intestinal compartments Intestinal compartments Duodenum Jejunum lleum

Control

Days of VCN treatment 5

266.8" 15.3 597.3" 35.4 642.8" 37.3

U

801.7 " 9.3 687.5" 86.9 607.3" 4.6

10

15

U

914.0U " 13.4 568.3" 29.7 1081.3U " 9.4

877.3 " 14.6 615.7" 11.6 1051.3U " 48

VCN was given orally in a dose of 2.7 mg kgy1 for the specified time intervals. Data are presented as mean " SEM Žrate of BrdU incorporation s the positively stained cells were counted microscopically per 40 villi in each compartment of small intestine .. Bromodeoxyuridine ŽBrdU. was injected i.p at a dose of 100 mg kgy1 1 h before killing mice. VCN-treated mice Ž n s 4. and control group Ž n s 8. killed after 10 days of starting the experiment. U Significantly different from control group at P- 0.05.

spleen lymphocytes and its local immunosupressive effect on the gut immune barrier. Our results illustrate that VCN decreases significantly the w 3 Hxthymidine uptake of PHA stimulated spleen T-lymphocytes only after 15 days of consecutive treatment, while its uptake was significantly inhibited after 5, 10 and 15 days post-VCN treatment in spleen T-lymphocytes stimulated by Con-A. Also, spleen B-lymphocytes stimulated with LPS showed a marked reduction in w 3 Hxthymidine uptake after VCN treatment for 5, 10 and 15 days. The inhibition of w 3 Hxthymidine uptake by spleen lymphocytes stimulated with Con-A, PHA or LPS, was used for the assessment of systemic immunotoxicity of many immunosuppressive agents, such as heavy metals Že.g. cadmium and mercury. in rats w17x; dimethylnitrosamine w22x and Monocrotaline w23x in mice. On the other hand, the difference in response of T-lymphocytes of VCN-treated animals to Con-A and PHA stimulation, may lighten a possible effect of VCN on T-lymphocyte maturation and differentiation w24, 25x. Spleen lymphocytes started to respond to Con-A stimulation after 5-days of VCN treatment, while they responded to PHA only following 15 days of treatment. T-cell dependent immunity plays a significant role in stabilizing the intestinal barrier and inhibits the bacterial translocation from GIT w26, 27x. Moreover, the number of lymphocytes in the intestinal lamina propria, Payer’s patches and mesenteric lymph node appears to be drastically incorporated in the bacterial translocation phenomenon w28, 29x. Also, it was reported that IgA, as a major antibody in the external secretions, is completely responsible for immune protection of mucosal surfaces and remains the best marker of local mucosal response w30, 31x. So it was useful to investigate the effect of VCN on IgA-producing cells in the different intestinal compartments. The results revealed the depletion in the number of IgA-producing cells in all intestinal compartments following VCN treatment and it was aggrevated with the duration of VCN administration. The inhibition of the number of IgA-producing cells in the gut was

considered as an immunosuppressive effect on humoral local gut immunity following treatment with some agents, e.g. cyclophosphamide w32, 33x and cyclosporine w34x. The results suggested that VCN induced a possible suppression on humoral immunity systemically Žby the decrease in mitogen response to LPS. and locally Žby the decrease in IgAproducing cells in GIT. as well as on cell mediated immunity Žby the inhibition of mitogen response to Con-A and PHA.. Also, the immune dysfunction was observed locally by the incapability of T-cells to cooperate with B-cells to produce IgA in the gut mucosa after VCN treatment. The local effect of VCN has been further verified by assessing the rate of cell proliferation in the gut of VCN-treated animals. Bromodeoxyuridine ŽBrdU. was used to detect S-phase cells through its incorporation in the newly synthesized DNA. The uptake of BrdU in the cell nucleus was conventionally used as a valuable guide for the study of prognosis in pre-cancer diseases and clinical oncology w35x. The results illustrated that the rate of cell proliferation was increased markedly in the duodenum following VCN treatment from 5 up to 15 days. The ileum compartment showed a significant increase in cell proliferation only after 10 and 15 days of VCNtreatment. These results are in accordance with several studies which reported that VCN increased the incidence of small intestinal tumors either in rats w12x or in workers w13, 36x exposed to VCN. Also, it was mentioned that chemical carcinogens may act by inducing mutations andror altering cellular proliferation w37, 38x. Furthermore, the overall risk for intestinal tumor induction is highly correlated with a significant level of DNA damage, a lower capacity of DNA repair and a marked increase in DNA synthesis over the constitutive level in the target cells w39, 40x. The investigators attributed the increase in DNA synthesis as to a regenerative effect for the chemically-induced intestinal injury. Different studies clarified the local effect of VCN on the intestinal cells and its gut DNA modifications w41]43x. It was found that VCN induces mucosal

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necrosis and ulcerogenic damage to the stomach and intestine w9, 10x. The induced necrosis was coupled with a marked decrease and depletion in gastric glutathione ŽGSH.. The depletion of GSH in the gastric mucosa allowed the exposure of nucleophilic sites in critical cellular macromolecules to the electrophilic action of VCN andror its metabolites and the extent of such exposure would be inversely proportional to the GSH concentration available. Moreover, the inherent relation between the immunotoxicity and the carcinogenic potential resulting in the gut tissues after chemical insult was reported w44x and different studies tightly linked the immunosuppression condition with the increase in carcinogenic potential of different compounds w45]48x. The present work illustrated that VCN induces a marked immunosuppessive effect systemically and locally on the gut, as well as increases the rate of cell proliferation of the gut mucosa. A matter which enabled us to propose a hypothesis for the mechanism of VCN immunotoxicity and its injurous effect of the gut mucosa. This hypothesis suggests that VCN causes a metabolic imbalance in intestinal tissues. The later includes depletion of the mucosal cells of its major defensive tool ŽGSH.. Depletion of GSH always results in perturbation of the redox mechanisms of the cells that leads to the abundance of reactive oxygen metabolites Žoxidative stress ., which tend to interact with GSH and enhances the metabolic imbalance together with disturbance of the redox cycle. This may cause damage to different cellular macromolecules. By this, the cascade of adverse cellular responses starts and ends in systemic and local immunosuppression as well as epithelial cell damage. The response of that damage is usually a regenerative increase in the rate of cell proliferation which in the presence of VCN Žor its reactive metabolites. may lead to mutations that ultimately may lead to tumorgenicity. The overall hypothesis might provide a possible immunological pathway of gut barrier failure and may find an explanation of why the gut is a target organ of VCN-induced carcinogenicity.

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