immunotoxicological assessment of methyl parathion in female b6c3f1 ...

IMMUNOTOXICOLOGICAL ASSESSMENT OF METHYL PARATHION IN FEMALE B6C3F1 MICE Patrick L. Crittenden Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi, USA

Russell Carr College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, USA

Stephen B. Pruett Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi, USA Methyl parathion is a widely used agricultural insecticide, and the recent unlicensed use of this compound in homes has led to the evacuation of approximately 1100 persons in Mississippi. Although the primary concern in such cases of acute exposure is neurotoxicity, a few organophosphorus compounds apparently have immunotoxic effects at dosages that do not produce neurotoxic symptoms. The purpose of the present study was to determine if this is the case for methyl parathion. Female B6C3F1 mice were exposed to methyl parathion by gavage, daily for 7, 14, 21, or 28 d ( at 6 mg/kg/ d) . Exposure for 14–28 d produced significant, dose-responsive inhibition of acetylcholinesterase ( the target molecule for methyl parathion-induced neurotoxicity) in brain or plasma, indicating that the compound was active. The following immunological parameters were evaluated: white blood cell counts and differentials, spleen and thymus weight and cellularity, splenic natural killer cell activity, nitrite production by peritoneal macrophages following activation in vitro, antibody response to sheep erythrocytes in vitro and in vivo, the cytotoxic T lymphocyte response to allogeneic tumor cells, and resistance to Streptococcus agalactiae and B16F10 melanoma cells. Methyl parathion at 1 or 3 mg/ kg/d significantly increased splenic natural killer cell activity. Nitrite production by macrophages was increased in mice treated with 1, 3, or 6 mg/ kg/d. The antibody response to sheep erythrocytes in vitro was significantly suppressed, but the humoral response to sheep erythrocytes in vivo was not affected. The cytotoxic Tlymphocyte response to allogeneic tumor cells was not significantly affected. Host resistance was not significantly decreased. Although it remains possible that immunological parameters not tested here may be affected by methyl parathion, the present results do not suggest substantial immunotoxic potential for this compound.

Received 23 July 1997; sent for revision 25 August 1997; accepted 29 September 1997. This work was supported by an Academic Research Enhancement Award from the National Institute of Environmental Health Sciences (ES05371) and by grant ES04394. S. B. Pruett is supported by a Research Career Development Award from National Institute of Alcohol Abuse and Alcoholism (K02 AA00201). Address correspondence to Stephen B. Pruett, Department of Cellular Biology and Anatomy, Louisiana State University School of Medicine, 1501 Kings Highway, Shreveport, LA 71130, USA. Email: [email protected] 1 Journal of Toxicology and Environmental Health, Part A, 54:1–20, 1998 Copyright © 1998 Taylor & Francis 0098-4108/98 $12.00 + .00

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P. L. CRITTENDEN ET AL.

Although the major effect of exposure of mammals to organophosphorus (OP) insecticides is neurotoxicity, some OP compounds are also immunotoxic. Several OP compounds have been evaluated for effects on one or more immunological parameters in rodent models. At neurotoxic dosages, parathion, malathion, O,O-dimethyl-O-2,2-dichlorovinyl phosphate (DDVP), sarin, tabun, and soman all significantly suppressed the antibody-forming cell response to the T-cell-dependent antigen, sheep erythrocytes (Casale et al., 1983, 1984; Clement, 1985). At nonneur otoxic dosages, some OP compounds are apparently immunosuppressive and others are not. In a preliminary report, Fan and colleagues (1978) noted that methyl parathion (3 mg/kg/d in the food) suppressed a number of important immunological parameters in mice. Similar effects were noted when rabbits received malathion (100 mg/kg/d) or DDVP (2.5 mg/kg/d) in the diet for several months (Desi et al., 1976). The most thoroughly evaluated OP with regard to immunotoxicity is O,O,S-trimethylphosphorothioate (O,O,S-TMP), a contaminant of a number of commercial pesticide preparations. This compound significantly suppresses a number of important immunological parameters at dosages that are not neurotoxic (Devens et al., 1985). In contrast, leptophos at dosages that significantly inhibit acetylcholinesterase does not suppress the generation of antibody-forming cells (Koller et al., 1976). Malathion at 0.1 LD50 per day does not decrease antibody or T-cell responses in mice (Rodgers et al., 1986), but it can increase release of histamine by mast cells (Rodgers et al., 1996). A recent thorough study failed to identify immunosuppressive effects of fenitrothion (Kunimatsu et al., 1996). These results indicate that some OP compounds can be immunosuppressive at nonneurotoxic dosages, but the available data are not sufficient to allow prediction of immunotoxicity on the basis of structural features. In the present study methyl parathion was evaluated for its effects on several important immunological parameters. The recent illegal use of methyl parathion as a pesticide in homes (it is licensed only for agricultural use) in Mississippi required numerous homes and businesses to be temporarily abandoned (Reid, 1996). Acute neurotoxic symptoms have been reported in some persons, suggesting substantial exposure. In addition, methyl parathion remains a widely used agricultural insecticide, and thousands of workers are exposed occupationally. The preliminary data of Fan and colleagues (1978) suggest that methyl parathion may be immunotoxic at subneurotoxic dosages. Therefore, the present study was conducted to evaluate the immunotoxicity of subneurotoxic dosages of methyl parathion. MATERIALS AND METHODS Animals Female C57Bl/6 × C3H F1 (B6C3F1) and DBA/2 mice, obtained through the National Cancer Institutes animal program, were quarantined

IMMUNOTOXICOLOGY OF METHYL PARATHION

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and allowed to recover from shipping-related stress for at least 1 wk prior to use. Animals were 7–10 wk of age at the beginning of all experiments. Animals were housed in a temperature (22–26°C) and humidity (40–60%) controlled environment on a 12-h light/dark cycle in an AAALAC-approved facility. All animals were given food (Purina lab chow) and water ad libitum. Sentinel mice were used to verify that the animal facilities were free of adventitious viruses and mycoplasma during the period of the study. Test Compound and Dosing Protocol Methyl parathion (purity >99%) was synthesized by Dr. Howard Chambers (Department of Entomology and Plant Pathology, Mississippi State University), as described previously (Chambers & Chambers, 1989). It was stored desiccated at –20°C until used. It was dissolved in foodgrade corn oil (vehicle) prior to treatment of animals. Methyl parathion was administered by gavage using a syringe and an 18-gauge stainless steel gavage needle. Methyl parathion was administered in a volume of 0.2 ml per mouse during the dose-response study. In all subsequent studies a volume of 0.1 ml per mouse was used. For vehicle control groups, 0.2 or 0.1 ml/mouse of corn oil was administered. Experimental Design The following experiments were done using methyl parathion: a doseresponse experiment, a time-course experiment, an experiment to assess the cytotoxic lymphocyte response, an experiment to assess the humoral immune response, an experiment to evaluate changes in thymocyte and splenocyte subpopulations, and two host resistance studies. The dose-response experiment included 5 groups (naive, vehicle, 1, 3, or 6 mg/kg/d) with 6 animals per group. Parameters measured were body weight, acetylcholinesterase (AChE) activity (brain, liver, plasma), hematology (leukocyte counts and differentials), thymus/spleen weight and cellularity, Mishell–Dutton antibody -forming culture (MD-AFC), nitrite production by peritoneal macrophages, and natural killer cell (NK) activity. Methyl parathion was administered daily for 28 d, and acetylcholinesterase activity and immunological parameters were evaluated 24 h after the last dose. The time-course experiment included 5 groups (naive, vehicle, and 6 mg/kg/d methyl parathion) with 5 animals per group. Parameters measured were AChE activity (brain), hematology (leukocyte counts), thymus/spleen weight and cellularity, and NK activity. During the time course the vehicle (corn oil) control group was treated daily for 28 d while the methyl parathion-treated (6 mg/kg) animals were treated for 7, 14, 21, or 28 d with methyl parathion. Treatment of animals with methyl parathion was initiated at different time points so all groups could be assayed on the same day. Acetylcholinesterase activity and immunological parameters were assayed 24 h after the last dose.

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Cell-mediated immunity was examined by measuring the lysis of 51Crlabeled P815 tumor cells in a 4-h culture. The design included 4 groups: vehicle, 1, 3, or 6 mg/kg/d. Each group included five mice. Vehicle and methyl parathion-treated mice were treated for 28 d. A cytotoxic T-lymphocyte response was induced by challenge with P815 tumor cells in vivo. All mice were injected with P815 cancer cells on d 20 of treatment. Twenty-four hours after the last dose of methyl parathion, all mice were euthanized (on d 29). Splenocytes from treated animals were cultured with 51Cr-labeled P815 cancer cells to determine cytotoxic T lymphocyte (CTL) activity as described later. Humoral immunity was examined by measuring the production of immunoglobulin M (IgM) and IgG antibodies specific for sheep erythrocytes (sRBC) (using an enzyme-linked immunosorbent assay described later). Two groups of animals were used (vehicle and 6 mg/kg/d of methyl parathion) with 5 mice per group. All animals were treated for 28 d. On d 16 prior to sacrifice all mice were immunized (intravenously) with sRBC. Retro-orbital bleeding of methoxyflurane anesthetized mice was performed on d 4 and 16 after immunization for optimal IgM and IgG production, respectively. Two host resistance models were employed for this study: the B16F10 melanoma model and Streptococcus agalactiae (group B streptococcus model, GBS). The methodological details for these models are described later; the experimental design follows here. Each model included 5 groups (naive, vehicle, 1, 3, or 6 mg/kg) with 8 animals per group. Vehicle control and methyl parathion-treated animals were treated for 28 d and naive controls were not treated during either study. Mice used in the B16F10 melanoma model were challenged with cancer cells on d 14 of treatment with methyl parathion. Methyl parathion treatment continued for 14 d following injection of cancer cells, and tumor growth was evaluated (as described later) 24 h after the last dose of methyl parathion. The animals used in the GBS model were immunized intraperitoneally with heat-killed GBS on d 14 and 21 of treatment with methyl parathion. Challenge of animals with live GBS occurred on d 29, 1 d after the last treatment with methyl parathion. Twenty hours postchallenge, samples of peritoneal fluid were obtained by peritoneal lavage, and bacteria were enumerated as described later. Statistical Analysis Means for vehicle control groups were compared to the mean for each treatment group using analysis of variance (ANOVA) followed by Dunnett’s test. All data sets were subjected to Bartlett’s test, and no significant differences in variances (indicating a violation of the assumptions of ANOVA) were identified. All statistical analyses were performed using the Instat software package (Graphpad Software, San Diego, CA).


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Immunological Assays Cell Counts and Hematology Mice were anesthetized using methoxyflurane and bled from the retro-orbital plexus. For isolation of serum, blood was collected into plastic tubes without anticoagulant. For white blood cell or red blood cell count, blood was collected into Becton-Dickinson minitubes with EDTA as anticoagulant. The mice were killed before recovering from anesthesia by placing them in a 100% CO2 atmosphere. Peritoneal cells were harvested by injecting 8 ml of ice-cold Dulbecco’s phosphate with 1% low endotoxin bovine serum albumin. The abdomen was then massaged, and the fluid was withdrawn. Spleen cells were obtained by pressing the spleens between the frosted ends of glass microscope slides that were wetted with RPMI 1640. Cell counts were done using an electronic cell counter (Coulter model Zf). Red blood cells were enumerated after appropriate dilution of an anticoagulated blood sample. White blood cell counts were done after lysing red blood cells with a commercial lysing reagent (SP manual hemoglobin and lysing reagent). Differential counts were done using Geimsa-stained blood smears. NK Cell Assay Natural killer cell activity of splenocytes was measured using a standard 51Cr-release assay with YAC-1 target cells, as described in our previous studies (Padgett et al., 1992; Pruett et al., 1992a). Data are expressed as lytic units per 107 splenocytes, and these values were calculated as described by Bryant and colleagues (1992). Nitrite Production by Peritoneal Macrophages Activated macrophages produce nitric oxide, which spontaneously reacts to form nitrite in culture. Nitrite production in response to stimulation by bacterial lipopolysaccharide (Sigma Chemical Co.), murine interferon-g (IFN-g , BoehringerMannheim), or both was evaluated using the Greiss reagent. The nitrite assay and the culture conditions used to activate the peritoneal macrophages have been described previously (Keil et al., 1995). Briefly, peritoneal macrophages were obtained by gavage and isolated by adherence to plastic. They were plated at 106/ml in 96-well culture plates and incubated with bacterial lipopolysaccharide (from Escherichia coli 0111:B4, Sigma Chemical Co.) (10 µg/ml), recombinant murine interferon-g (IFN-g ) (1000 U/ml), or both for 24 h. Nitrite concentration in culture supernatants was measured using the Greiss reagent. Mishell–Dutton Antibody Forming Cell Cultures The Mishell– Dutton culture system (Mishell & Dutton, 1967) was used to evaluate the ability of spleen cells from methyl parathion-treated mice to respond to a T-dependent antigen (sheep erythrocytes, sRBC). Antibody-forming cells (AFC) were measured using a plaque assay. The culture conditions and the plaque assay have been described previously (Pruett et al., 1992a). Assessment of Cytotoxic T-Cell Activity Cytotoxic T-cell function was measured following immunization of mice with allogeneic P815 mastocytoma cells. This was done essentially as described by Kerkvliet

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and colleagues (1990). Briefly, mice were immunized by intraperitoneal injection of 10 7 viable P815 cells. Ten days after immunization, the spleens were removed, single-cell suspensions were prepared, and cytotoxic T-cell activity was measured using a 4-h 51Cr-release assay with labeled P815 target cells. Lytic units per 10 7 spleen cells were calculated as described by Bryant and colleagues (1992). In Vivo Antibody Response to sRBC The antibody response to sRBC was evaluated as described previously (Pruett et al., 1992a) by immunizing mice intravenously with intact sRBC (2 × 108/mouse), then analyzing serum samples for IgM and IgG specific for sRBC using an enzyme-linked immunosorbent assay (ELISA). The antigen used to coat plates was a high salt extract prepared from washed sheep erythrocyte membranes by the method of van Loveren et al. (1991). Plates were coated by incubating this extract at 0.1 µg protein/well in phosphate-buffered saline (pH 7.4) overnight at 4°C. Mice were bled on d 4 and d 16 following immunization, and the d-4 sample was analyzed for IgM anti-sRBC antibodies. The d-16 sample was analyzed for IgG anti-sRBC antibodies as described previously (Pruett et al., 1992a). These time points were selected because peak or near-peak responses occur at these times (Carson & Pruett, 1996; Pruett et al., 1992a). Host Resistance Assays All mice used in the B16F10 melanoma model were challenged by intravenous injection of 1 × 105 viable B16F10 melanoma cells (kindly provided by Dr. Albert E. Munson, National Institute for Occupational Safety and Health). Twenty-four hours after the last treatment with methyl parathion, all animals were euthanized and both lungs were removed. Lungs were fixed with Bouin’s solution and the numbers of externally visible tumor nodules were counted using a dissecting microscope. This procedure has been used by the National Toxicology Program to evaluate immunotoxicity of a number of compounds (Luster et al., 1988). The animals used in the Streptococcus agalactiae (group B streptococcus, GBS) model were immunized intraperitoneally with heat-killed GBS. The minimal dose of heat-killed bacteria required to provide ~90% protection of mice from challenge with a lethal dose of live bacteria was determined in preliminary experiments, as described previously (Barnes et al., 1992). Animals were challenged with live GBS 1 d after the last treatment with methyl parathion. Twenty hours postchallenge, samples of peritoneal fluid were obtained by peritoneal lavage. Serial dilutions of the lavage fluid were plated on Mueller–Hinton agar, and the number of viable cells was determined by counting the number of colonies. The strain of GBS used in these experiments was selected for its virulence in mice. The LD50 is less than 20 bacteria per mouse (Barnes et al., 1992). This indicates that mice have essentially no innate resistance to this organism, and must develop protective acquired immune responses to survive the challenge dose used in these studies (~100 bacteria per mouse) (Barnes et al., 1992). We have shown previously that the number


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of bacteria in the peritoneum at 20 h postchallenge correlates well to survival time in this model (Barnes et al., 1992). Assay for Acetylcholinesterase Activity Acetylcholinesterase activity in plasma, cerebral cortex, and medulla oblongata was assayed using a modification of the method of Ellman described by Chambers et al. (1988). Cerebral cortex, medulla oblongata, and plasma samples were isolated from euthanized animals and frozen at –70°C until the assay was performed. All tissues (except plasma) were homogenized in 0.008 M sucrose (cerebral cortex and medulla oblongata). For cerebral cortex and medulla oblongata AChE, 100 µl of homogenate was added to 4 ml of Tris-HCl buffer (AChE buffer). For the AChE assay, sample tubes were prepared in triplicate. In 2 of the 3 tubes, vehicle (ethanol) was added, and the third received 0.001 M eserine sulfate, a specific inhibitor of AChE. Each tube was vortexed and placed in a 37°C shaking water bath for 15 min to allow for temperature equilibrium of the enzyme and to allow eserine sulfate to inhibit AChE in the blank. The reaction was initiated by the addition of 40 µl of 0.1 M acetylthiocholine to each tube at 10-s intervals. Tubes were vortexed and returned to the 37°C shaking water bath for 15 min. The reaction was terminated by the addition of 500 µl of a 4:1 solution of 5% sodium dodecyl sulfate:0.024 M 5,5’-dithio-bis(2nitrobenzoic acid) to each tube at 10-s intervals, vortexed, and the contents poured into plastic cuvettes. The absorbance was measured at 412 nm using a spectrophotometer. The absorbance of the eserine sulfate blank for each sample was subtracted to correct for non-AChE-mediated hydrolysis. Quantification of protein content in tissues was done using the Folin phenol reagent with bovine serum albumin (BSA) as a standard (Lowry et al., 1951). RESULTS Body Weight Change The change in body weight was measured as an indicator of generalized toxicity in methyl parathion treated animals. The animals receiving 3 mg/kg/d methyl parathion had significantly greater body weight gain than the vehicle control (Table 1). The body weight changes from the doseresponse study are representative of body weight changes seen in other experiments. No decrements in body weight gain were observed in methyl parathion-treated mice in any experiment. Acetylcholinesterase Activity Acetylcholinesterase activity was measured to confirm the biological activity of methyl parathion in vivo. Acetylcholinesterase activity was significantly inhibited in blood plasma at a dosage of 6 mg/kg/d (Figure 1). Acetylcholinesterase activities in two regions of the brain were also measured. Acetylcholinesterase activity was significantly inhibited in

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TABLE 1. Changes in body weight during a 28-d course of daily methyl parathion administration Group

n

Increase in body weight (g) ± SE

Naive Vehicle 1 mg/kg/d 3 mg/kg/d 6 mg/kg/d

6 6 5 5 6

2.07 ± 1.52 ± 2.28 ± 3.37 ± 1.91 ±

a

0.293 0.277 0.251 0.088a 0.081

Significant at p < .05 as compared to the vehicle control group.

cerebral cortex and medulla oblongata with dosages of 3 and 6 mg/kg/d (Figure 1). The effect of methyl parathion (6 mg/kg/d) on brain acetylcholinesterase was examined at various time points. Methyl parathion significantly inhibited cerebral cortex acetylcholinesterase activity after 21 or 28 d of dosing, but not after 7 or 14 d. Medulla oblongata acetylcholinesterase was inhibited at the 14, 21, and 28 d time points (data not shown). Hematological Parameters Methyl parathion did not cause significant changes in differential leukocyte counts (Figure 2). In an initial experiment, significant decreases in blood leukocyte counts were noted in methyl parathion-treated mice (data not shown). However, in three subsequent experiments, the number of peripheral white blood cells was not affected by methyl parathion (Figure 2). Thymus and Spleen Weight and Cellularity Thymus weight was not significantly different from vehicle control values in methyl parathion-treated mice (data not shown). Thymus cellularity was significantly decreased in mice treated with methyl parathion at 1 mg/kg/d, but not in mice treated with methyl parathion at 3 or 6 mg/kg/d (Figure 3A). Thymus weight was not significantly affected at any time point during administration of methyl parathion at 6 mg/kg/d (data not shown). Thymus cellularity was decreased significantly at the 7-d time point in methyl parathion-treated animals (Figure 3B). Thymus cellularity was not significantly different from control values at other time points. Spleen weights in methyl parathion-treated animals were not significantly different from vehicle control values at any dosage of methyl parathion (data not shown). Spleen cellularity was significantly decreased in the animals treated with methyl parathion at 6 mg/kg/d as compared to vehicle control (Figure 3C). Spleen cellularity was significantly increased in animals treated with methyl parathion at 6 mg/kg/d for 7 d, 14 d, or 21 d (Figure 3D). Spleen cellularity after 28 d of dosing was not significantly different from that of the vehicle control group (Figure 3D).


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Antibody Production Splenocytes from methyl parathion-treated mice produced significantly fewer antibody-forming cells (AFC) upon challenge with sRBC in Mishell– Dutton cultures (Figure 4A). This decrease was dose dependent. The production of IgM and IgG antibodies in vivo in response to immunization with sheep erythrocytes was assayed by ELISA. Animals treated with 6 mg/kg/d of methyl parathion had no decrements in serum IgM or IgG antibodies to sRBC when measured at the optimum time for these responses (Figure 4B).

FIGURE 1. Acetylcholinesterase specific activity in animals treated with methyl parathion at dosages of 1, 3, or 6 mg/kg/d for 28 d. Values shown represent the mean activity ± SE for groups of five mice. Statistical significance was determined by ANOVA followed by Dunnett’s t-test, and values significantly different from those of the vehicle control are indicated by asterisk (p £ .05).

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FIGURE 2. White blood cell counts and differential cell counts in methyl parathion-treated mice. Each panel represents results from an independent experiment. In (C), the indicated dosages of methyl parathion were given daily for 28 d, and differential counts were done on d 29. Values shown represent means ± SE. None of the differences shown are significant (by ANOVA, n = 5 mice per group). FIGURE 3. Thymus and spleen cellularity in mice treated with methyl parathion. (A and C) Mice were treated daily with methyl parathion at the indicated dosages for 28 d. (B and D) Mice were treated with methyl parathion at 6 mg/kg/d for the indicated periods of time. Values shown represent means ± SE for groups of five mice. Statistical significance was determined by ANOVA followed by Dunnett’s t-test, and values significantly different from those of the vehicle control are indicated by asterisk (p £ .05).


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FIGURE 4. Effect of methyl parathion on the antibody response to sheep erythrocytes. (A) Number of antibody-forming cells (AFC) produced following in vitro challenge with sRBC in Mishell–Dutton cultures. (B) Results of a separate experiment in which mice were immunized with sRBC during treatment with methyl parathion and bled for determination of IgM 4 d after immunization and for determination of IgG 16 d after immunization. Antibody concentrations were evaluated by ELISA. Values shown represent means ± SE for groups of five mice. Statistical significance was determined by ANOVA followed by Dunnett’s t-test, and values significantly different from those of the vehicle control are indicated by asterisk (p £ .05).


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Natural Killer Cell Activity NK cell activity was increased in mice treated with methyl parathion at 1 or 3 mg/kg/d in comparison to the vehicle controls. Lysis of target cells, expressed as lytic units, was more than twofold greater in these groups than in vehicle controls (Figure 5A). NK cell activity was not sig-

FIGURE 5. Natural killer cell activity in methyl parathion-treated mice. (A) Mice were treated daily with methyl parathion at the indicated dosages for 28 d. (B) Mice were treated with methyl parathion at 6 mg/kg/d for the indicated periods of time. Splenic NK cell activity was measured 24 h after the last dose of methyl parathion. Values shown are means ± SE for groups of five mice. Significance was determined by ANOVA followed by Dunnett’s t-test, and values significantly different from those of the vehicle control are indicated by asterisk (p £ .05).

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nificantly altered during the time-course experiment, in which methyl parathion was administered at 6 mg/kg/d. No significant changes were observed in NK cell activity at any time point (Figure 5B). Nitrite Production by Peritoneal Macrophages Methyl parathion treatment significantly increased nitrite production by peritoneal macrophages stimulated in vitro with IFN-g , lipopolysaccharide (LPS), or IFN-g and LPS. Interferon- g -stimulated macrophages obtained from mice exposed to methyl parathion produced significantly more nitrite than macrophages from vehicle or naive control mice. Macrophages obtained from methyl parathion-treated mice produced significantly more nitrite in response to in vitro stimulation with LPS than control. Nitrite production by peritoneal macrophages stimulated with IFN-g and LPS in combination was significantly higher in macrophages from methyl parathion-treated mice than in those from vehicle control mice (Figure 6). Cytotoxic T Lymphocyte Activity Cytotoxic T lymphocyte (CTL) activity was induced in vivo by immunization with viable P815 (allogeneic) tumor cells and was measured using 51 Cr-labeled P815 tumor cells in an in vitro assay. CTL activity (expressed as lytic units) was not significantly different from vehicle control levels at any dosage of methyl parathion evaluated in this study (Figure 7).

FIGURE 6. Nitrite production by macrophages in methyl parathion-treated mice. Mice were treated with methyl parathion at the indicated dosages for 28 d. Twenty-four hours after the last dose, peritoneal macrophages were stimulated with IFN-g , LPS, or IFN-g and LPS for 24 h in vitro. The value for LPS at 3 mg/kg/d is missing because of experimental error. Values shown represent means ± SE for groups of five mice. Statistical significance was determined by ANOVA followed by Dunnett’s t-test, and values significantly different from those of the vehicle control are indicated by asterisk (p £ .05).


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FIGURE 7. Cytotoxic T lymphocyte (CTL) activity in methyl parathion-treated mice. Mice were immunized with allogeneic P815 mastocytoma on d 20 of a 28-d course of methyl parathion treatment. Twenty-four hours after the last dose of methyl parathion (on d 29), splenic CTL activity was measured using a standard 51 Cr-release assay. This allowed assessment of CTL activity at the optimum time point (10 d). Values shown represent means ± SE. No differences are significant (by ANOVA, n = 5 mice per group).

Resistance to B16F10 Tumor Cells The ability of methyl parathion to alter host resistance to cancer cells in vivo was examined utilizing B16F10 melanoma cells. Animals were injected with B16F10 cells intravenously, and tumor nodules on the lungs were counted on d 15. No statistically significant differences were noted between control and methyl parathion-treated animals at any dosage of methyl parathion (Figure 8). Thymus weight and cellularity were also measured in this experiment, and there were no significant differences between groups (data not shown). Resistance to Group B Streptococci The ability of methyl parathion to alter host resistance to GBS in vivo was examined. Vehicle control and methyl parathion-treated animals (1, 3, or 6 mg/kg/d) were immunized on d 14 and 21 with heat-killed GBS. Preliminary experiments demonstrated that 106 heat-killed bacteria given at these time points protected ~90% of mice from a lethal dose (100 viable bacteria) of GBS. On d 29 all animals (naive, vehicle, and methyl parathion-treated) were challenged intraperitoneally with pathogenic GBS. Peritoneal lavage was performed 20 h after challenge, and lavage fluid was used to make pour plates to determine the number of live GBS. Previous studies have shown that the number of live GBS 20 h after challenge is strongly inversely correlated with survival (Barnes et al., 1992).

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FIGURE 8. Resistance to B16F10 melanoma cells in methyl parathion-treated mice. Mice were treated with methyl parathion for 28 d. B16F10 melanoma cells were administered intravenously on d 13 of methyl parathion administration. Values shown are means ± SE for number of tumor nodules on both lungs. No differences are significant (by ANOVA, n = 5 mice per group).

No significant decreases in host resistance (i.e., increases in numbers of bacteria) were observed in methyl parathion-treated animals, all of which were immunized with heat-killed GBS during administration of methyl parathion (Figure 9). Nonimmunized control mice had high levels of GBS in the peritoneum, demonstrating the requirement for immunization to induce resistance to these bacteria. DISCUSSION A preliminary report (abstract) has been published suggesting that methyl parathion exerts a number of immunosuppressive effects in mice (Fan et al., 1978). In addition, minor effects on a few immunological parameters have been noted in another study (Street & Sharma, 1975). However, a relatively complete characterization had not been done prior to the present study. Using several components of the National Toxicology Program (NTP) tier system (Luster et al., 1988), the immunotoxic effects of methyl parathion in vivo were investigated in B6C3F1 mice. In contrast to the other reports, the data reported here do not suggest that methyl parathion is immunotoxic. We and others have found that methyl parathion and other OP compounds can be immunotoxic if administered at dosages that produce overt signs of neurotoxicity (Casale et al., 1984; Pruett et al., 1992b). However, these effects could be secondary to the stress response induced by high dosages of OP compounds (Kunimatsu et al., 1996). In the present study, the dosages were sufficient to doseresponsively decrease AChE activity in the brain and serum, but not sufficient to produce overt signs of neurotoxicity.


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Methyl parathion did not consistently affect peripheral blood leukocyte numbers or differential counts. Although significant decreases in leukocyte numbers were noted in one experiment, these were not dose responsive and did not occur in any other experiment. Therefore, it seems unlikely that decreased leukocyte counts are a consistent, important outcome of methyl parathion exposure. Thymus weight and cellularity were examined after 28 d of treatment with methyl parathion. At most time points and most dosages there were no significant effects. However, thymus cellularity was significantly decreased after treatment with methyl parathion at 1 mg/kg/d for 28 d. Methyl parathion at 6 mg/kg/d for 7 d also significantly decreased thymus cellularity. Because the observed changes were small and not dose or time dependent, it seems unlikely that they are important immunologically. Spleen cellularity, but not spleen weight, was affected by administration of methyl parathion. Methyl parathion at 6 mg/kg/d administered for 28 d significantly decreased (~20%) spleen cellularity. In a second experiment, spleen cellularity was increased ~20% by treatment with 6 mg/kg/d of methyl parathion at the 7-, 14-, and 21-d time points. However, spleen cellularity at the 28-d time point was not significantly affected. Although methyl parathion has been reported to decrease spleen weight and cellularity (Fan et al., 1978), the present study does not consistently support this observation. Furthermore, the impact of such minor changes as those noted here on the immune system is uncertain.

FIGURE 9. Resistance to Streptococcus agalactiae in methyl parathion-treated mice. All mice except the nonimmunized (non-imm.) control group were immunized with heat-killed Streptococcus agalactiae twice during the course of methyl parathion administration. The control group was immunized and treated with vehicle instead of methyl parathion. The methyl parathion-treated groups were also immunized. Mice were challenged intraperitoneally with a lethal dose of live streptococci (100 colony-forming units), and the number of bacteria per peritoneum was evaluated at 24 h after administration of bacteria. Values shown represent means ± SE for groups of five mice. Statistical significance was determined by ANOVA followed by Dunnett’s t-test, and values significantly different from those of the control group are indicated by asterisk (p £ .05).

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Administration of methyl parathion in vivo decreased the primary humoral immune response (plaque-forming cells, PFC) to sRBC in vitro. Decreases occurred in a dose-responsive manner, and the number of plaques observed in cultures from mice treated with 6 mg/kg/d of methyl parathion was less than 50% of vehicle control values. However, decreases in PFC responses in Mishell–Dutton cultures may not always reflect in vivo antibody responses in animals treated with immunotoxicants. To examine in vivo antibody production an ELISA was used to measure serum IgM and IgG production in response to immunization with sRBC in vivo. No significant differences between vehicle- and methyl parathiontreated mice were observed. The results of the in vivo antibody response are probably more meaningful than the results from Mishell–Dutton cultures in predicting the effect of methyl parathion on host resistance. These data are not in agreement with a report of decreased antibody production to Salmonella typhimurium challenge in Swiss (ICR) mice exposed to methyl parathion (Fan et al., 1978). This could be related to differences in the models (e.g., different mouse strain, different methods of dosing). Because natural killer cells and CTL are thought to constitute the major immune parameters involved in resistance to neoplasia and the intracellular phase of viral infection (Luster et al., 1988), these parameters were evaluated in methyl parathion-treated mice. Methyl parathion at 1 or 3 mg/kg/d for 28 d significantly increased NK activity, but a dosage of 6 mg/kg/d did not affect NK activity at any time point. The enhancement of NK cell activity by inducers such as polyinosinic polycytidylic acid or a virus infection improves the protective effect of these cells (Daniels et al., 1987). Therefore, it is conceivable that the lower dosages of methyl parathion used in this study could improve host resistance to some microbes. The effect of methyl parathion on CTL activity was examined, and no significant differences in CTL activity were observed at any dose of methyl parathion. Considering that methyl parathion does not decrease NK or CTL activity, one would not expect host resistance to tumor or viral challenge to be affected by methyl parathion exposure. Peritoneal macrophage activity, measured by nitrite production in vitro, was significantly increased in macrophages from methyl parathiontreated animals. However, it is not known if these relatively small changes would have any impact on innate or acquired resistance to microbes. The production of other reactive intermediates (O2– and H2O2) and the antigen presentation by peritoneal macrophages in methyl parathion-treated animals have not been examined. However, the data obtained here do not suggest that methyl parathion suppresses macrophage activation. Two models were used to examine the ability of methyl parathion to alter host resistance. The ability of methyl parathion to alter host resistance to cancer cells was examined using the B16F10 melanoma model. Treatment with methyl parathion had no effect on host resistance to cancer cells. The absence of methyl parathion-induced changes in host resis-


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tance is consistent with the absence of decreases in NK and CTL activity in methyl parathion-treated mice, because these are major defense mechanisms against cancer cells (Luster et al., 1988). Methyl parathion has been reported (in an abstract) to decrease host resistance to Salmonella typhimurium in Swiss (ICR) mice (Fan et al., 1978). However, in the present study, host resistance to GBS in vivo was not affected by treatment with methyl parathion. This result is consistent with the lack of changes observed in peritoneal macrophage activity and serum antibody production, because these are the major determinants of host defense against these bacteria (Stanton et al., 1981). The basis for the difference in the results of Fan et al. (1978) and those reported here is not clear, but the use of different bacteria or different mouse strains may be involved. In summary, methyl parathion affected some immunological parameters at dosages that did not cause overt neurotoxicity. Inhibition of acetylcholinesterase activity confirmed that methyl parathion was biologically active. Immune-system parameters were not consistently decreased by treatment with methyl parathion. Host resistance to B16F10 cells or GBS in vivo was not significantly affected by methyl parathion. Therefore, data from the present study suggest that the immune system is not seriously compromised by subneurotoxic dosages of methyl parathion. It remains possible that increases in natural killer cell activity and macrophagederived nitrites could indicate immune dysregulation that might have adverse consequences. REFERENCES Barnes, D. B., Hardin, J. M., and Pruett, S. B. 1992. Acute infection of mice with highly virulent group B streptococci as a host resistance model for immunotoxicity assessment. Arch. Toxicol. 66:423–429. Bryant, J., Day, R., Whiteside, T. L., and Herberman, R. B. 1992. Calculation of lytic units for the expression of cell-mediated cytotoxicity. J. Immunol. Methods 146:91–103. Carson, E., and Pruett, S. B. 1996. Development and characterization of a binge drinking model in mice for evaluation of the immunological effects of ethanol. Alcoholism Clin. Exp. Res. 20:132– 138. Casale, G. P., Cohen, S. D., and DiCapua, R. A. 1983. The effects of organophosphate-induced cholinergic stimulation on the antibody response to sheep erythrocytes in inbred mice. Toxicol. Appl. Pharmacol. 68:198–205. Casale, G. P., Cohen, S. D., and DiCapua, R. A. 1984. Parathion-induced suppression of humoral immunity in inbred mice. Toxicol. Lett. 23:239–247. Chambers, J. E., and Chambers, H. W. 1989. An investigation of acetylcholinesterase inhibition and aging and choline acetyltransferase activity following a high level acute exposure to paraoxon. Pestic. Biochem. Physiol. 33:125–131. Chambers, J. E., Wiygul, S. M., Harkness, J. E., and Chambers, H. W. 1988. Effects of acute paroxon and atropine exposures on retention of shuttle avoidance behavior in rats. Neurosci. Res. Commun. 3:85–92. Clement, J. G. 1985. Hormonal consequences of organophosphate poisoning. Fundam. Appl. Toxicol. 5:S61–S67. Daniels, M. J., Menache, M. G., Burleson, G. R., Graham, J. A., and Selgrade, M. K. 1987. Effects of

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NiCl2 and CdCl2 on susceptibility to murine cytomegalovirus and virus-augmented natural killer cell and interferon responses. Fundam. Appl. Toxicol. 8:443–453. Desi, I., Farkas, I., Varga, G., Gonczi, C., Szlobodnyik, J., and Kneffel, Z. 1976. Immunosuppressive effects of hydrocarbon and organophosphate pesticide administration. Egeszegtudomany 20: 358–368. Devens, B. H., Grayson, M. H., Imamura, T., and Rodgers, K. E. 1985. O,O,S-Trimethyl phosphorothioate effects on immunocompetence. Pestic. Biochem. Physiol. 24:251–259. Fan, A., Street, J. C., and Nelson, R. M. 1978. Immunosuppression in mice administered methyl parathion and carbofuran by diet. Toxicol. Appl. Pharmacol. 45:235. Keil, D. E., Luebke, R. W., and Pruett, S. B. 1995. Differences in the effects of dexamethasone on macrophage nitrite production: Dependence on exposure regimen (in vivo or in vitro) and activation stimuli. Int. J. Immunopharmacol. 17:157–166. Kerkvliet, N. I., Baecher-Steppan, L., Smith, B. B., Youngberg, J. A., Henderson, M. C., and Buhler, D. R. 1990. Role of the Ah locus in suppression of cytotoxic T lymphocyte activity by halogenated aromatic hydrocarbons (PCBs and TCDD): Structure-activity relationships and effects in C57Bl/6 mice congenic at the Ah locus. Fundam. Appl. Toxicol. 14:532–541. Koller, L. D., Exon, J. H., and Roan, J. G. 1976. Immunological surveillance and toxicity in mice exposed to the organophosphate, leptophos. Environ. Res. 12:238–242. Kunimatsu, T., Kamita, Y., Isobe, N., and Kawasaki, H. 1996. Immunotoxicological insignificance of fenitrothion in mice and rats. Fundam. Appl. Toxicol. 33:246–253. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275. Luster, M. I., Munson, A. E., Thomas, P. T., Holsapple, M. P., Fenters, J. D., White, K. L., Lauer, L. D., Germolec, D. R., Rosenthal, G. J., and Dean, J. H. 1988. Methods evaluation. Development of a testing battery to assess chemical-induced immunotoxicity: National Toxicology Program’s guidelines for immunotoxicity evaluation in mice. Fundam. Appl. Toxicol. 10:2–19. Mishell, R. I., and Dutton, R. W. 1967. Immunization of dissociated mouse spleen cell culture from normal mice. J. Exp. Med. 126:423–442. Padgett, E. L., Barnes, D. B., and Pruett, S. B. 1992. Disparate effects of representative dithiocarbamates on selected immunological parameters in vivo and cell survival in vitro in female B6C3F1 mice. J. Toxicol. Environ. Health 37:559–571. Pruett, S. B., Barnes, D. B., Han, Y.-C., and Munson, A. E. 1992a. Immunotoxicological characteristics of sodium methyldithiocarbamate. Fundam. Appl. Toxicol. 18:40–47. Pruett, S. B., Han, Y., Munson, A. E., and Fuchs, B. A. 1992b. Assessment of cholinergic influences on a primary humoral immune respone. Immunology 77:428–435. Reid, B. 24 November 1996. EPA knew about pesticide problem. Clarion Ledger, Jackson, Mississippi, 1A, 13A. Rodgers, K., Amand, K. S., and Xiong, S. 1996. Effects of malathion on humoral immunity and macrophage function in mast cell-deficient mice. Fundam. Appl. Toxicol. 31:252–258. Rodgers, K. E., Leung, N., Ware, C. F., Devens, B. H., and Imamura, T. 1986. Lack of immunosuppressive effects of acute and subacute administration of malathion on murine cellular and humoral immune responses. Pestic. Biochem. Physiol. 25:358–365. Stanton, B. F., Baltimore, R. S., and Shedd, D. G. 1981. Effects of route and time of administration of antiserum on protection of mice from lethal infection due to group B Streptococcus Type III. Infect. Immun. 31:391–395. Street, J. C., and Sharma, R. P. 1975. Alteration of induced cellular and humoral immune responses by pesticides and chemicals of environmental concern: Quantitative studies of immunosuppression by DDT, Aroclor 1254, carbaryl, carbofuran, and methylparathion. Toxicol. Appl. Pharmacol. 32:587–602. van Loveren, H., Verlaan, A. P. J., and Vos, J. G. 1991. An enzyme-linked immunosorbent assay of antisheep red blood cell antibodies of the classes M, G, and A in the rat. Int. J. Immunopharmacol. 13:689–695.