Effects of Aspirin-Like Drugs on Mitogen- Stimulated DNA Synthesis of Lymphocytes from Pregnant Rats and Offspring

Home , Salicylamide

003 1-3998/88/2301-0093$02.00/0 PEDIATRIC RESEARCH Vol. 23, No. 1, 1988 Copyright O 1988 International Pediatric Research Foundation, Inc. Printed in U.S.A.

RICHARD M. MILLIS, MICHAEL C. EWII, GODWIN U. OFFIAH, BARBARA P. HYDE, AND ENID M. KNIGHT Animal Physiology Laboratory, Department of Zoology, Graduate School ofArts and Sciences and Department of Human Nutrition and Food, School of Human Ecology, Howard University, Washington, D.C. 20059

ABSTRACT'. Previous studies have shown that salicylates suppression of human lymphocytes (1-4). However, results of in and protein-calorie malnutrition compromise immunologi- vivo studies of salicylates (aspirin) and suppression of lymphocyte cal responses in humans and experimental animals. The transformation have been very conflicting. present study compared the effects of prenatal normal and Crout et al. (5)found marked suppression of blastogenesis in low protein diets, with and without aspirin-like drug treat- lymphocyte transformation studies using PHA and PWM on ments, on lymphocyte blastogenesis measured by tritiated blood samples obtained from normal volunteers taking three thymidine uptake for DNA synthesis in splenic lympho- aspirin tablets (totaling 900 mg) five times daily for 4 days cytes from pregnant rats and their offspring following (therapeutic dosage). stimulation with the mitogens concanavalin A, phytohe- On the other hand, studies by Smith et al. (6) have shown that magglutinin, and pokeweedmitogen. Aspirin treatment was when 12 healthy subjects were placed on a dose of 600 mg aspirin associated with increased lvm~hocvte-- - thvmidine u~takefor five times/day for 14 days, the capability of their lymphocytes to blastogenesis in pregnant rats fed the normal protein con- incorporate [3H]thymidine in response to PHA and allogenic trol diet and their offspring. The phytohemagglutinin-stim- lymphocytes was not statistically different from that of non- ulated increase detected in offspring lymphocytes could not treated control subjects. The authors concluded that, within a 2- be statistically guaranteed. A low protein diet alone and a wk period, aspirin administered orally at therapeutic doses does normal protein diet combined with salicylamide treatment not suppress lymphocyte responsiveness to either PHA or allo- was associated with decreased blastogenesis in pregnant genic lymphocytes. Egorin et al. (7) obtained similar results in rats but not in their offspring. Salicylamide or aspirin treating human subjects with 600 or 900 mg aspirin four times combined with a low-protein diet decreased blastogenesis daily for 1 wk. They found that when taken orally, salicylate did in both dams and their offspring. Aspirin combined with a not induce any irreversible change in the capability of lympho- normal protein diet did not adversely affect blastogenesis cytes to respond to isolectin stimulation. in either pregnant rats or their offspring. This study sug- Although these results on lymphocyte reaction to mitogens are gests that low dietary protein and aspirin-like drugs may conflicting, the possibility remains that treatment with aspirin- independently decrease lymphocyte blastogenesis of preg- like drugs at a reduced dietary protein level might decrease the nant rats and in combination they may also reduce lym- immunocompetence of pregnant mothers and their offspring. phocyte blastogenesis in offspring. The significance of When used in pregnancy above pharmacological levels, aspirin- increased lymphocyte blastogenesis in both mothers and like drugs might therefore pose a risk to the fetus of mothers offspring following aspirin treatment of pregnant rats fed with poor nutritional status. a normal protein diet is unclear. (Pediatr Res 23: 93-97, Previous studies have shown that maternal protein restriction 1988) in rats retards placental and fetal growth (8). The importance of maternal dietary protein to human fetal growth and development Abbreviations has been recently underscored by directive of the U.S. Food and Drug Administration to increase the RDA of protein for pregnant PHA, phytohemagglutinin mothers. Offspring of mothers who ingest salicylates chronically PWM, pokeweed mitogen have had significantly reduced birth weights and increased peri- Con A, concanavalin A natal mortality (9). The negative nitrogen balance, uncoupling RDA, Recommended Dietary Allowance of oxidative phosphorylation, and adrenocortical activation as- HBSS, Hanks' balanced salt solution sociated with salicylate treatment (10) might produce conditions MASH, Multiple Automated Sample Harvester unfavorable to lymphocyte proliferation and antibody produc- SPSS, Statistical Package for the Social Sciences tion. The purpose of this study is to examine the interaction effect of a protein-deficient diet and aspirin-like drug ingestion during pregnancy on maternal and offspring lymphocyte transformation The results of in vitro studies on the effect of aspirin have led in response to the mitogens PHA, PWM, and Con A. some researchers to assume that aspirin ingestion leads to in vivo METHODS Received December 22, 1986; accepted September 22, 1987. Correspondence and requests for reprints Richard M. Millis, Ph.D., Assistant Professor, The Experiment I. Animals. One hundred twenty pregnant Spra- American University, 4400 Massachusetts Avenue N.W., Biology Department/ gue-Dawley rats 3 to 4 months old were used in this study. Hurst Hall 101, Washington, D.C. 2001 6. Nonpregnant weights ranged from 200 to 300 g prior to distri- 94 MILLIS ET AL. bution to cages. They were bred with normal male rats at the offspring from experiment I, group 1 were designated as group 7 Howard University Animal Resources Facility in Beltsville, MD. in experiment 11, etc. (Table 1). Diets and Food Intake. Rats were divided into six weight- Each group in experiment I1 was fed a normal control diet of matched groups of 20 rats each. Table 1 summarizes the dietary 25% protein ad libitum for 60 days. Weight gain was recorded treatments of control and experimental groups. To determine once a week. At the end of day 60, animals were sacrificed and the effects of aspirin-like drugs on an important determinant of the methods and procedures performed during experiment I were immune responses of pregnant rats and their offspring, experi- followed. mental groups of pregnant rats were fed either aspirin or salicyl- Statistical significance of differences between control and ex- amide. Animals were fed ad libitum from day 1 of gestation perimental groups was guaranteed by Duncan's multiple range through delivery (days 19-2 1). Weight gain and food intake were test computed electronically by the SPSS software program on recorded twice weekly. an IBM 270 computer. After delivery, five pups from each group were culled to each dam, and a total of 10 dams from each group was used for RESULTS weaning. The remaining 10 dams from each group were sacrificed by cervical dislocation and serum from blood samples was Efffcts of aspirin on Con A-stimulated lymphocytes. Aspirin collected and frozen for subsequent analysis. treatment of pregnant rats fed the normal protein control diet Mitogenic Response of Splenic Lymphocytes. Each spleen was (group 2) was associated with increased amounts of radioactive removed with sterile scissors and forceps, weighed, and trans- thymidine incorporated into DNA within lymphocytes stimu- ferred to 10 ml of cold (5" C) HBSS (Biofluid Inc., Rockville, lated by the mitogen Con A; aspirin treatment of pregnant rats MD), which was later transferred to a sterile glass Petri dish for fed the low protein diet (group 5) was associated with additional cell suspension preparation. decreases in thymidine uptake compared to animals on the Suspensions were washed twice by centrifugation with 10 cc normal protein diet both with (group 2) and without aspirin HBSS at 1500 rpm for 10 min at 5" C in a refrigerated centrifuge. treatment (group 1) (Fig. 1). The resulting pellet was suspended in 2 ml of cold culture media Similarly, Con A-stimulated lymphocytes from offspring of consisting of 9 1 % RPMI media, 4% antibiotic (250,000 U pen- dams fed aspirin with the normal protein control diet exhibited icillin/streptomycin), and 5% fetal calf serum. greater blast transformation than lymphocytes from offspring of Acetic acid (3%) was used to lyse erythrocytes and the suspen- dams fed the normal protein diet without aspirin. Lymphocytes sion was transferred to a white cell hemocytometer for counting. from the offspring of dams fed aspirin with the low-protein diet The entire cell suspension was diluted to 5 x 10' cells/ml of showed significantly less blastogenesis than lymphocytes from culture media. From this dilution, 5 x lo5 cells in 0.1 ml were offspring of dams fed aspirin with the normal protein diet and cultured in triplicate in microtiter culture dishes with either PHA less blastogenesis than lymphocytes from offspring of dams fed 0.3 13 pg in 0.1 ml; Con A 2.5 pg in 0. l ml; and PWM in a ratio the low protein diet without aspirin (Fig. 1). of 1:20 pg in 0.1 ml. Control wells with unstimulated cells Effects of aspirin on PHA-stimulated lymphocytes. When stim- contained 5 x lo6 cells in 0.1 ml culture medium. ulated by PHA, lymphocytes from pregnant dams treated with The cells were incubated for 72 h in 5% C02 atmosphere at aspirin and fed both the normal and the low protein diet (groups 37" C. Five h prior to the termination of incubation, each well 2 and 5) exhibited significantly greater blast transformation than was labeled with 1.0 @Ci[3H]thymidine (New England Nuclear, lymphocytes from pregnant dams fed the normal protein diet Boston, MA) and reincubated under the same conditions. At the without aspirin treatment (group 1) (Fig. 2). PHA-stimulated end of the incubation period, the cells were harvested via a blast transformation of lymphocytes from dams fed the low- MASH (Microbiological Associates, Walkersville, MD) onto protein diet with aspirin (group 5) was greater than that of dams MASH I1 glass fiber filters and allowed to dry overnight. fed the low protein diet without aspirin treatment (group 4). The following day, each section of the filter was individually PHA-stimulated lymphocytes from offspring showed no blas- placed in 5 ml of liquid scintillation cocktail consisting of 0.1 % togenic effects following maternal aspirin treatment under either of 1,4-bis-2-5-phenyloxazolylbenzenescintillation grade and 5% normal or low protein dietary conditions (Fig. 2). 2,5-diphenyloxazole scintillation grade in counting vials and Effects of aspirin on PWM-stimulated lymphocytes. PWM- allowed to stand in the dark for 30 min, after which the samples stimulated lymphocytes from pregnant rats exhibited greater were placed in a liquid scintillation counter. Results obtained blast transformation when normal dietary protein with aspirin after counting were expressed in counts per minute (cpm). (group 2) was compared to normal dietary protein conditions Experiment 11. This study was designed to determine if treat- without aspirin treatment (group I) (Fig. 3). ment of the dams affected the offspring, specifically to determine PWM-stimulated blast transformation of lymphocytes from whether salicylamide, aspirin, or dietary protein affects the im- pregnant rats treated with aspirin and fed a low protein diet mune system of the offspring. (group 5) was significantly less than that from pregnant rats fed Animals. Sixty male and female Sprague-Dawley weanling rats, the normal protein diet with aspirin treatment (group 2), but age 3 wk, were used. Their weights ranged from 24 to 60 g prior transformation of lymphocytes from group 5 rats fed the low to distribution to cages. They were divided into six groups of 20 protein diet combined with aspirin was greater than that from rats each, corresponding to the groups in experiment I, i.e. pregnant rats fed the low protein diet without aspirin (group 4).

Table 1. Dietary treatment ofnrou~s Group Experiment I Experiment I1 Dietary protein (%) Drug Normal protein Control 1 Experimental 2 0.25% aspirin Experimental 3 I % salicylamide

Low protein Control 4 10 8 Experimental 5 11 8 0.25% aspirin Experimental 6 12 8 I % salicylamide ASPIRIN-LIKE DRUGS AND PREGNANCY

Con A PHA b

Fig. 2. Phytohemagglutinin response of dams and offspring lympho- Fig. 1. Con A response of dams and offspring lymphocytes. Vertical cytes. Vertical bars show mean + SEM tritiated thymidine uptake (cpm X lo3) of splenic lymphocytes from pregnant rats (groups 1-6) and bars show mean + SEM tritiated thymidine uptake (cpm X 10') of splenic lymphocytes from pregnant rats (groups 1-6) and offspring offspring (groups 7-12) following mitogenic stimulation with PHA. (groups 7-12) following mitogenic stimulation with Con A. Group 1-6 Group 1-6 (experiment I, Table 1) lymphocytes were taken from preg- (experiment I, Table 1) lymphocytes were taken from pregnant rats fed nant rats fed the following diets: 1. normal (25%) protein control; 2. the following diets: I. normal (25%) protein control; 2. normal (25%) normal (25%) protein with 0.25% aspirin added; 3. normal (25%)protein protein with 0.25% aspirin added; 3. normal (25%) protein with 1% with 1% salicylamide added; 4. low (8%) protein control; 5. low (8%) salicylamide added; 4. low (8%) protein control; 5. low (8%) protein with protein with 0.25% aspirin added; 6. low (8%) protein with 1% salicyl- 0.25% aspirin added; 6. low (8%) protein with I% salicylamide added. amide added. Group 7-12 (experiment 11, Table 1) lymphocytes were Group 7-12 (experiment 11, Table I) lymphocytes were taken from taken from offspring of the pregnant rats fed diets 1-6, respectively. offspring of the pregnant rats fed diets 1-6, respectively. Offspring were Offspring were 3-wk-old weanlings fed the normal (25%) protein control 3-wk-old weanlings fed the normal (25%) protein control diet for 60 diet for 60 days. Equivalent letters above each bar, indicate homogeneous days. Equivalent letters above each bar indicate homogeneous subsets, subsets, differences between which were determined not to be statistically differences between which were determined not to be statistically signif- significant (p > 0.05); nonequivalent letters indicate heterogeneous sub- icant (p > 0.05); nonequivalent letters indicate heterogeneous subsets, sets, differences between which were determined to be statistically signif- differences between which were determined to be statistically significant icant (p < 0.05) by Duncan's analysis of variance. Values on ordinate (p < 0.05) by Duncan's analysis of variance. Values on ordinate expressed expressed in cpm x lo3. in cpm x lo3. the normal protein control diet without aspirin (group 7). How- ever, lymphocytes from group l l offspring fed the low protein However, blastogenesis in PWM-stimulated lymphocytes from diet with aspirin showed less blast transformation than either group 5 pregnant rats fed the low protein diet with aspirin was those from group 8 offspring of dams fed aspirin with the normal not significantly different than that from pregnant rats fed the protein diet or group 10 offspring from dams fed the low-protein normal control diet without aspirin (group 1). diet without aspirin treatment. Figure 3 shows that PWM-stimulated lymphocytes from off- Efects of salicylamide on Con A-stimulated lymphocytes. spring of dams fed aspirin with both the normal protein diet Splenic lymphocytes from pregnant rats fed the normal and low- (group 8) and the low protein diet (group 11) exhibited greater protein diets with salicylamide (groups 3 and 6) exhibited de- blastogenesis than lymphocytes from the offspring of dams fed creased blastogenesis following stimulation by Con A (Fig. 1). MILLIS ET AL. PWM and offspring of dams fed the normal protein diet with salicyl-

1501 Tb amide (Fig. 1). Effects of salicylamide on PHA-stimulated lymphocytes. PHA stimulation of lymphocytes from pregnant rats fed the normal protein diet with salicylamide showed decreased blast transformation. Increased blastogenesis was detected in lymphocytes from dams fed the low protein as compared with the normal protein diet combined with salicylamide treatment. Salicylamide treatment of pregnant rats fed the low protein diet was associated with a lower level of PHA-stimulated blastogenesis than in pregnant rats fed the normal protein control diet without salicylamide, but with a higher level than in either those fed the normal protein diet with salicylamide or fed the low protein diet without salicylamide (Fig. 2). Salicylamide had no effect on blast transformation of PHA- stimulated lymphocytes from offspring of dams fed both normal and low protein diets (Fig. 2). Effects of salicylamide on PWM-stimulated lymphocytes. Compared to controls (group I), blastogenesis was lower in PWM-stimulated lymphocytes from pregnant rats fed the normal protein diet with salicylamide (group 3) but greater in those from dams fed the low-protein diet with salicylamide (group 6) (Fig. 3). Blastogenesis in the latter group 6 animals was even greater than that detected in PWM-stimulated lymphocytes from pregnant rats fed either the low protein diet without salicylamide (group 4) or the normal protein diet with salicylamide (groups 3 and 1). Figure 3 shows that salicylamide treatments of dams fed both the low-protein and normal protein diets were associated with increased blastogenesis of PWM-stimulated lymphocytes from their offspring (groups 9 and 12) compared to that in lymphocytes from offspring of dams fed either the normal protein diet or the low-protein diet without salicylamide treatment (groups 7 and 10).

DISCUSSION Acetylsalicylic acid (aspirin) is one of the most commonly used drugs because of its efficacy as an analgesic, antipyretic, and antiinflammatory agent, the low cost and relative safety. Al- though there is no evidence that therapeutic doses of salicylates Fig. 3. PWM response of dams and offspring lymphocytes. Verlical cause fetal damage in humans, infants born to women who bars show meal1 + SEM tritiated thymidine uptake (cpm x lo3) of chronically ingest salicylates may have significantly decreased splenic lymphocytes from pregnant rats (groups 1-6) and offspring birth weights (10). The present studies suggest that maternal and (groups 7-12) following mitogenic stimulation with PWM. Group 1-6 neonatal effects on in vitro lymphocyte blastogenesis may be (experiment I, Table I) lymphocytes were taken from pregnant rats fed observed when chronic maternal ingestion of aspirin-like drugs the following diets: 1. normal (25%) protein control; 2. normal (25%) is associated with protein-deficient diets. It is probable that the protein with 0.25% aspirin added; 3. normal (25%) protein with 1% low protein maternal diet decreased the serum protein concen- salicylamide added; 4. low (8%)protein control; 5. low (8%)protein with tration that would have had the effect of decreasing the amount 0.25% aspirin added; 6. low (8%)protein with 1% salicylamide added. of aspirin-like drug bound to serum albumin and increasing the Group 7-12 (experiment 11, Table 1) lymphocytes were taken from amount of unbound, pharmacologically active drug, relative to offspring of the pregnant rats fed diets 1-6, respectively. Offspring were the control normal maternal dietary protein conditions. How- 3-wk-old weanlings fed the normal (25%) protein control diet for 60 ever, the extent to which variable concentrations of active aspirin- days. Equivalent Ieclers above each bar indicate homogeneous subsets, like drugs may have become incorporated into the in vitro differences between which were determined not to be statistically signif- lymphocyte blast transformation test system when applied to icant (p > 0.05); nonequivalent letlers indicate heterogenous subsets, lymphocytes from pregnant rats under normal versus low protein differences between which were determined to be statistically significant conditions was not determined. (p< 0.05) by Duncan's analysis of variance. Values on ordinate expressed Numerous investigations have focused on the effects of aspirin in cpm x lo3. on various aspects of cellular immunology. In vitro studies by several researchers have demonstrated that aspirin (salicylates) Salicylamide treatment of pregnant rats fed the low-protein diet suppresses lymphocyte transformation (1-3). Aspirin added to (group 6) was associated with significantly less Con A-stimulated cultures of normal lymphocytes inhibited blastogenic response lymphocyte blastogenesis than that in pregnant rats fed the low of these cells to crude preparations of PHA (1, 2, 4). protein diet without salicylamide treatment (group 4). Crout et al. (5) reported a blastogenic response ratio after in Increased Con A-stimulated lymphocyte blastogenesis was de- vivo salicylate ingestion of 0.49, compared with ratios of 0.25 (1) tected in lymphocytes from offspring of dams fed the normal and 0.16 (2) in cultures exposed simultaneously to PHA and protein diet with salicylamide. Low dietary protein with salicyl- salicylate in vitro. The authors concluded that oral administration amide treatment of dams was associated with decreased Con A- of aspirin significantly suppresses lymphocyte transformation. stimulated blastogenesis in offspring compared to that in both Ganter and Zuckner (3) suggest that ingestion of a single thera- offspring of dams fed the low-protein diet without salicylamide peutic dose of aspirin by a normal person can completely inhibit ASPIRIN-LIKE DRUGS AND PREGNANCY

in vitro lymphocyte transformation by PHA for periods of 25 to SUMMARY AND CONCLUSIONS 36 h. In pregnant dams, lymphocytes stimulated by Con A, PHA, Mitogen-stimulated splenic lymphocyte proliferation was uni- and PWM exhibited increased blastogenesis in vitro in response formly decreased in pregnant rats fed the normal protein diet to aspirin treatment combined with the normal protein diet; plus salicylamide and mimicked the effects of the low protein under low protein dietary conditions, increased in vitro blasto- diet on the dams. Similarly, the effects of salicylamide and aspirin genesis was observed only in response to PHA stimulation. Only in the offspring of rats fed the normal protein diet mimicked the Con A-stimulated lymphocytes from pregnant dams exhibited effects of low maternal dietary protein on the offspring of dams very low levels of in vitro blastogenesis with aspirin treatment fed the normal protein diet; increased splenic lymphocyte blas- under low dietary protein conditions. Con A- and PWM-stimu- togenesis resulted from activation by the mitogens Con A and lated lymphocytes from offspring of dams fed the normal protein PWM, with no effect seen in PHA-stimulated lymphocytes. diet with aspirin showed increased in vitro blastogenesis that was Mitogen-stimulated splenic lymphocyte proliferation was uni- not observed when lymphocytes were stimulated by PHA. In the formly increased in pregnant rats fed the normal protein diet offspring, decreased in vitro lymphocyte blastogenesis associated with aspirin treatment. These results suggest that aspirin treat- with aspirin treatment of dams was clearly detected only under ment might increase cellular and humoral immunity in pregnant Con A stimulation of lymphocytes from dams treated with low rats under normal dietary protein conditions, but these findings dietary protein condition during gestation. are in sharp contrast to the generally accepted result that in vivo Only Con A-stimulated lymphocytes exhibited uniformly de- aspirin treatment decreases in vitro mitogen-stimulated lympho- creased in vitro blastogenic responsiveness from both pregnant cyte blastogenesis. Indeed, such conflicting results emphasize the rats and their offspring as a result of treatment with salicylamide. problem of applying the significance of in vitro findings to in Because of the problems of equating in vitro with significant vivo cellular immune responsiveness. in vivo immunological deficiency, the statistically significant in The increase in PWM-stimulated lymphocyte blastogenesis vitro lymphocyte blastogenic effects described herein should be associated with aspirin treatment of pregnant dams under normal interpreted cautiously pending confirmation by tests of in vivo dietary protein conditions might suggest a tendency toward immune functions. increased tumorigenesis because of greater B cell mediated synthesis of blocking antibodies (1 1, 12). However, greater control Acknowledgment. The authors acknowledge the assistance of of Walker neoplasm growth attributed to aspirin might actually Doris Hughes, D.V.M. who supervised breeding of animals used result from synthesis of an antiosteolytic factor (13) and may in this study at the Howard University Animal Resource Facility, therefore be unrelated to immune function per se. Beltsville, MD. A more recent report that enhancement of suppressor T cell activity was associated with decreased antibody secretion in REFERENCES response to blood lymphocyte activation by PWM (14) suggests I. Opelz G, Terasaki PI 1973 Suppression of lymphocyte transformation by that the combination of conditions that produces decreased aspirin. Lancet 2:478-480 PWM-stimulated lymphocyte proliferation associated with in- 2. Pachman LM, Esterly NB, Peterson RD 1971 The effect of salicylate on the creased Con A-stimulated lymphocyte proliferation may provide metabolism of normal and stimulated human lymphocytes in vitro. 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Berger EM, Harada RN, Vatter AE, Bowman MC, Repine JE 1984 Cyclical ciated with no effect on PHA activation in offspring of dams fed abnormalities in the bactericidal function, superoxide production, and lyso- a low protein diet with and without treatment with the aspirin- zyme activity of neutrophils obtained from a healthy woman during men- like drugs, salicylate and salicylamide, in this study. struation: reversal by pretreatment with aspirin. J Infect Dis 149:413-419 Erratum Volume 22, Number 5, November 1987, pp. 6 16-620. Atriovenous Nodal Function in the Immature Canine Heart, by Jorge McCormack, Henry Geland, Hui Zu, Juan Villafane, Adrienne Stolfi, and Arthur S. Pickoff. The above article is reprinted in its entirety on pages 99-103 due to a printer's error. AV was incorrectly written out as 'atriovenous' rather than 'atrioventricular.' Both the printer and the publisher regret this error.