Parathion-induced suppression of humoral immunity in inbred mice

239

Toxicology Letters, 23 (1984) 239-247 Elsevier

TOXLett. 1313

PARATHION-INDUCED INBRED MICE* (Immunosu~pression;

SUPPRESSION

organophosp~ate

OF HUMORAL

IMMUNITY

IN

insecticides; Jerne plaque assay)

GEORGE P. CASALE**, STEVEN D. COHEN and RICHARD A. DiCAPUA Section of Pharmacology and Toxicoiogy, School of Pharmacy, University of Connecticut, Storrs, CT &&S3 (U.S.A.) (Received April 18th, 1984) (Revision received June 15th, 1984) (Accepted August lst, 1984)

SUMMARY Numbers of IgM plaque-forming cells (PFC) were reduced by 65% in C57B1/6 mice given parathion 2 days after immunization with sheep red blood cells (SRBC). The immunosuppressive dose (16 m&/kg, p.o.) caused signs of cholinergic poisoning and 20% mortahty. Survivors appeared to have recovered fully from the cholinergic crisis at the time of immunologic assay. However, these animals had reduced tissue cholinesterase (ChE) activities and decreased numbers of nucleated spleen cells. Immunosuppression was apparent on day 4 but not on day 3 or days 5-8 of the primary IgM response. Reduction of serum hemagglutinin titers coincided with reduction of the number of splenic PFC. A lower dose of insecticide (4 mg/kg, p.o.) did not produce signs of poisoning and was not immunosuppressive. The number of I-day IgG PFC was reduced by 45% when parathion (16 mg/kg, p.o.) was given 6 days after immuni~tion, but not when parathion was given 2 days after immuni~tion. The data suggest that cholinergic stimulation and/or the associated toxic chemical stress may be involved in parathion-induced immunosuppression.

*Presented, in part, at the 21st annual meeting of the Society of Toxicology, February 22-26, 1982, Boston, (U.S.A.). **Recipient of NRSA award No. ES05205 in environments toxicology from the National Institute of Environmenta Health Sciences. Abbreviations: ANOVA, analysis of variance; ChE, cholinesterase; DDVP, dichlorvos; MEM, Minimal Essential Media; OP, organophosphate; PFC, plaque-forming cells; SRBC, sheep red blood cells. 0378-4274/84/$ 03.00 @ Elsevier Science Publishers B.V.

240

INTRODUCTION

Laboratory studies have demonstrated altered humoral immune responses in animals exposed to Op insecticides [l-4]. A recent study [5] demonstrated reduced numbers of IgM PFC in mice exposed to a single dose of parathion, malathion, or DDVP. Immunosuppression occurred after an insecticide dose which produced cholinergic signs, but the occurrence and degree of immunosuppression did not correlate with a particular time-response profile of insecticide-induced cholinesterase inhibition. Duration and time of onset of OP-induced immunosuppression were not determined. Consequently it is possible that insecticide exposure may have delayed rather than suppressed the immunologic response. In this study, we demonstrated that parathion exposure produced a short-lived suppression of the primary IgM response to,SRBC, and, that an appropriately timed acute exposure to parathion reduced numbers of &day IgG PFC. METHODS

Male inbred (C57Bl/6) mice (Charles River Mouse Farms, Wilmington, MA), 7-10 weeks of age, were housed 10 per cage and allowed free access to food (Purina Laboratory Chow) and water. Mice were kept in air-conditioned (25°C) animal quarters on a 12-h (7-7) light-dark cycle. Cages were stainless steel and had a grid flooring suspended 2.5 inches above waste pans containing sawdust. Mice were immunized by a single tail vein injection of 0.1 ml of SRBC (20% v/v) in balanced salt solution. All SRBC were collected from the same ewe (Department of Animal Sciences, College of Agriculture) and were stored in sterile Alsever’s solution [6] for 2-4 weeks at 4°C before use. Parathion, 99.2% purity (Stauffer Chemical Co.), was mixed with corn oil to provide appropriate doses in a volume of 5 ml/kg, p.o. Immunized controls were given corn oil. Mice were decapitated and selected organs were removed and processed immediately. Brains and livers were homogenized at 0-4°C in 0.1 M phosphate buffer, pH 8.0. Blood was collected in heparinized tubes and separated by centrifugation. Erythrocytes (RBC) were washed once with 0.9% saline before storage and were lysed with distilled water immediately before assay. Brain and liver homogenates, undiluted plasma and washed RBC were stored at -2O’C until assayed for cholinesterase activity. Spleens were dissociated into single-cell suspensions in MEM and were processed for enumeration of IgM and IgG PFC, as described below. Spleen cellularity was determined by counting viable (trypan blue excluding) nucleated cells in suspension. Tissue ChE activity was measured by a modification of the method of Ellman et al. [7] described previously by Casale et al. [5]. In all cases, tissue and substrate concentrations were selected to provide optimum conditions at which tissue enzyme activity was rate-limiting.

241

IgM PFC were assayed by the method of Jerne et al. [8] as modified by Plotz et al. [9]. IgG PFC were developed in the presence of enhancing antibody [ 10, 111. All counts were made with duplicate slides and grouped data were expressed as geometric means. Hemagglutinins were measured by the microtitration method of Sever [12] and grouped data were expressed as geometric means of individual serum titers. Serological and PFC data were transformed to logarithms [lo] and analyzed by one-way or two-way ANOVA [ 131. Untransformed spleen cellularity, spleen-body weight ratio and ChE data were analyzed by one-way ANOVA. RESULTS

Effects of acute parathion exposure on the primary IgM response Splenic PFC and serum hemagglutinins were quantified at several time points during a primary immune response of mice treated with corn oil or parathion. At the beginning of experimentation, age-matched mice were randomly distributed into two groups and then immunized with SRBC. In experiment Ia, one group received a single dose of corn oil (5 ml/kg) and the other a single dose of parathion (16 mg/kg), 2 days after immunization. In an earlier study [5], this treatment schedule produced a considerable reduction (80%) in the number of 4-day IgM PFC. Immunologic responses were assessed on days 3 through 8, after SRBC. Those animals treated with parathion displayed signs of cholinergic poisoning 1 to 2 h after treatment. Signs of poisoning were severe at 3 to 5 h when most of the observed mortalities (20%) occurred. Animals surviving to the time of immunologic assay appeared to have recovered fully from poisoning, but these animals had reduced brain ChE activity throughout the period of immunologic assessment (Table I). On day 4, mice exposed to insecticide had significantly fewer PFC (Fig. 1) and reduced titers of serum hemagglutinins (Fig. 2) in comparison to corn oil-treated mice. No significant immunologic changes were observed, on the remaining days of the response. Decay of the IgM response was clearly evident by day 8, in both control and insecticide-treated mice (Fig. 1). On day 4 when numbers of splenic PFC were reduced, insecticide-treated mice had lower spleen:body weight ratios and decreased numbers of nucleated spleen cells (Table II). Reduced spleen:body weight ratios were first observed on day 3 (1 day after parathion). Both spleen:body weight ratio and number of nucleated spleen cells returned to control values by day 5 and remained so on day 8. As expected, the number of nucleated cells per spleen was increased in the immunized control mice, on response days 3 and 4, in comparison to other response days (Table II). In experiment Ib, mice were dosed with 4 mg parathion/kg or 5 ml corn oil/kg, 2 days after immunization. Immunological responses and tissue ChE were assayed on day 4, after SRBC. OP-treated mice exhibited no visible signs of toxicity. Brain and liver ChE activities in these animals were normal, whereas plasma activity was

242

TABLE

I

CHOLINESTERASE Ex~.~

Ia

ACTIVITY

IN TISSUES

OF PARATHION-DOSED

Parathion

Day of assay

Cholinesterase

(mgikg)

(post SRBC)

(olo control)c

activityb

Brain

Plasma

4

*50*4

N.D.d

96+5

16

6

*56+2

N.D.

113+5 107*3

16

8

*67+5

N.D.

4

4

104+3

*69-+3

IIa

16

8

*72&3

*83+5

IIb

16

8

*58+3

*53 * 1

“All animals

were immunized

oil, on day 2 (Exp.

cholinesterase and IIb).

Liver

16

lb

or corn

MICE

activities

with sheep red blood

were quantified,

along

bX + S.E. where N z 5. Values significantly ‘X* S.E. for control hydrolyzed/min/g

activities

brain

cells (SRBC),

Ia, Ib, IIa) or day 6 (Exp.

IIb).

from control

98~5 *87+3

on day 0, then treated On the indicated

with IgM PFC (Exp.

different

95*4

days

with parathion of assay,

tissue

Ia and Ib) or IgG PFC (Exp. IIa

at Ps 0.05 are noted with an asterisk.

(N = 10) were 10.4 i 0.2, 6.9 + 0.2, 3.4 + 0.2 and 1.6 f 0.2 amol substrate

or liver, or per ml plasma

or packed

RBC,

respectively.

dNot determined.

reduced (Table I). The OP treatment did not alter number of PFC (not shown), spleen:body weight ratio or spleen cellularity (Table II). Effects

of acute parathion exposure on the primary IgG response

The IgG phase of the primary response to SRBC was evaluated, after a single dose of insecticide. Animals were treated with parathion (16 mg/kg) 2 days (Exp. IIa) or

0

7

4

3

Days

After

8

Immunization

Fig. 1. IgM-PFC time profile in immunized mice, after a single dose of parathion (16 mg/kg p.0.). Mice were dosed with corn oil (- - -) or parathion (a - - ) 2 days after immunization with SRBC, and killed at the indicated times. (PC 0.05) determined

Each point is a mean by one-way ANOVA.

of 5 or more

animals.

Bars indicate

S.E.

*Significance

243

~ __~. ~~

_

-

4

3 Days

. 6

5 After

7

6

Immunization

Fig. 2. Time course of serum hemagglutinin titers in immunized mice, after a single dose of parathion (16 mg/kg p.0.). Mice were dosed with corn oil (- --) or parathion ( - * -) 2 days after immunization with SRBC and killed at the indicated times. Each point is the mean of 5 or more animals. Bars indicate S.E. *Significance (P
TABLE II SPLEEN-BODY MICE Exp.’

WEIGHT RATIOS AND SPLEEN CELLULARITIES Parathion (mglkg)

Ia

0 16 0 16 0 16 0 16

OF PARATHION-DOSED

Day of assay (post SRBC)

bSpleen x 10’ B.W.

3

4.39Zto.11 *3.69+0.14 3.93io.14 ‘3.52kO.14 3.65 kO.33 3.58kO.13 3.88kO.36 4.OOf 0.28

83.71 k5.12 102.40+5.73 *13.57 + 2.52 67.80+ 6.65 69.36 + 2.70 71.00+5.33 62.93 f 3.42

4 5 8

bViable cells

x 10-6

Spleen 90.60 + 9.96

Ib

0 4

4

3.11f0.14 3.60*0.09

86.3 +5.3 89.5 k8.3

iIa

0 16

8

3.09*0.12 3.07+0.09

69.1 k4.0 66.6 k2.8

IIb

0 16

8

3.34kO.06 *3.07 & 0.08

18.6 +4.2 +63.0 f4.7

aAll animals were immunized with SRBC, on day 0, then treated with parathion or corn oil, on day 2 (Exp. Ia, Ib, IIa) or day 6 (Exp. IIb). On the indicated days of assay, spleen:body weight ratios and numbers of viable cells were determined, along with numbers of IgM PFC (Exp. Ia and Ib) or IgG PFC (IIa and IIb). bFt S.E. where NZ 5. Values which differ significantly from the vehicle at P< 0.05 are noted with an asterisk.

244

6 days (Exp. IIb) after immunization. Mortality rates were 24% and 39070,respectively. Survivors were killed on day 8 of their response to SRBC. On the day of assay, all survivors appeared healthy and exhibited no signs of poisoning. With the exception of liver in experiment IIa, tissue ChE activities were significantly depressed after either treatment (Table I, Exp. IIa and IIb). Spleen:body weight ratio and spleen cellularity were reduced 2 days after parathion (Table II: Exp. IIb) but were normal 6 days after parathion (Table II. Exp. IIa). Eight-day IgG PFC were decreased only in those animals dosed with insecticide 6 days after immunization and killed 2 days later (Fig. 3: Exp. IIb). DISCUSSION

In this study, we have shown that parathion treatment suppressed the primary IgM response to SRBC, in mice. PFC of control and treated mice were assayed daily, during a period in which the bulk of primary, IgM PFC were produced. Four-day PFC of treated mice were reduced to 35% of controls. This result confirmed previous reports [3, 51. However, in contrast to the present study, the latter evaluated pesticide effects at one time point (day 4) during the primary immune response to SRBC. Consequently, there remained a possibility that reduced numbers of 4-day PFC indicated a shift in the time course rather than suppression of the primary response. As our data indicate, the time profile of IgM PFC was indeed shifted, after OP exposure. The peak of the response occurred on day 5 rather than PFCXI~~/

Spleen

Itb Experiment Fig. 3. Effect

of parathion on the IgG response to SRBC. Mice were dosed with 16 mg parathion/kg (P< 0.10) determined 2 days (Ha) or 6 days (IIb) after SRBC, and were killed on day 8. *Significance by two-way ANOVA (N > 5 mice per group).

245

day 4. However, this peak was indistinguishable from the number of 5-day PFC on the descending limb of the control response. In fact, PFC responses of control and treated mice were indistinguishable on all but day 4 of the response period we examined. However, during response days 3 through 8, parathion-treated mice produced considerably fewer PFC in relation to the total number of PFC produced by control mice. This difference was not due to an indiscriminate loss of cells from the spleen, since the number of IgM PFC expressed per IO6 nucleated spleen cells was reduced by 49% (not shown). The transient nature of the OP-induced depression in numbers of splenic PFC might suggest a temporary relocation of PFC to a tissue site(s) other than spleen. This appears unlikely in view of the reduction in titer of serum hemagglutinins after pesticide exposure. Our data indicate both a change in the time course and a suppression of the primary IgM response to SRBC, as a consequence of acute exposure to parathion. Although the mechanism of immunosuppression remains to be determined, there are a number of possible mechanisms which merit close consideration. In general, parathion and/or its metabolites may have acted directly or indirectly on the immune system. More specifically, since acetylcholine receptors are present on lymphocytes and monocytes [ 14,151, cholinergic stimulation, consequent to OP exposure, may have altered immunologic function [5]. Alternatively, OP-induced toxic chemical stress may have produced a corticosteroid-dependent immunosuppression. Szot and Murphy [16] have demonstrated increased serum concentrations of corticosterone in rats treated with sublethal doses of parathion. The immunosuppressive effects of corticosteroids are well known [17-,9]. Finally, parathion may have had a short-lived, direct effect on the immune systeJ& Such an effect could be a result of OP inhibition of cell-associated esterases [20, 211. Both the ability of OPs to inhibit esterases [22] and the existence of membrane-associated esterases of leukocytes [23, 241 are well documented. Our data, concerned with parathion-induced effects on the number of primary IgG PFC, are consistent with each of the mechanisms discussed above. Parathion was given 2 days or 6 days after immunization, then, PFC were assayed on day 8 (after SRBC). The number of PFC was reduced only in those mice given the pesticide 6 days after SRBC (i.e., 2 days before ass,ay). OP-mediated effects would appear to be short-lived, as was the case for the IgM response. In summary, parathion exposures, which caused signs of cholinergic poisoning, produced a short-lived suppression of the primary IgM response to SRBC and reduced the number of S-day IgG PFC. Additional studies are required to determine whether parathion-induced immunologic effects are a consequence of (1) OPinduced increases in concentrations of tissue acetylcholine which may act on leukocyte-associated acetylcholine receptors, (2) OP-induced increases in concentrations of tissue corticosteroids which may impair leukocyte function, and/or (3) a direct action on leukocytes, e.g., inhibition of leukocyte-associated esterases. If OPinduced immunosuppression is the effect of severe cholinergic stimulation, im-

246

munologic impairment may be of consequence in severely poisoned individuals. Alternatively, OP insecticides may act directly on the immune system. This possible outcome would warrant long-term low-dose studies of the effects of these compounds on immunologic functions and resistance to disease. ACKNOWLEDGEMENTS

This research was supported by Grant No. ES02524 from the National Institute of Environmental Health Sciences and BRSG No. RR 05743. The authors wish to thank Dr. Jack Dean for his helpful advice; C. Cowan, C. Heninger, W. Duggan, S. Patierno and S. Moore for valuable technical assistance and Dr. J. Glaz for assistance with the statistical evaluation of the data. Parathion was kindly provided by Stauffer Chemical Co. REFERENCES

10 11

12 13 14

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