Evaluation of suppressive effects of paraquat on innate immunity in Balb/c mice

Abstract

The toxic effect of paraquat (PQ), an herbicide that has been used widely in agriculture, on some parameters of the immune system was investigated. PQ was administered to Balb/c mice as intraperitoneal doses of 0.01, 0.1, or 1.0 mg PQ/kg body weight (BW) for a total of 28 days. Besides the histopathological examination of each host’s vital organs, measures of their splenic and bone marrow cellularity, blood macrophage/granulocyte phagocytic activity, total serum immunoglobulin M (IgM) and IgG, complement C3, and splenic CD49b (natural killer) cell levels, as well as of serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase and complement-mediated lytic activity (CH₅₀), activities, were then performed following the respective final treatments. The results indicate that high and medium doses of PQ (i.e., 1 and 0.1 mg/kg) could lead to suppressed phagocytic activity by circulating macrophages/granulocytes. The data also revealed that the high PQ dose induced a significant decrease (p < 0.05) in spleen cellularity and splenic CD49b cell levels, along with numerous histopathological changes in the spleen. However, at none of the doses tested did PQ produce changes in serum levels of C3, total IgG or IgM, or in the CH₅₀. At 0.01 mg PQ/kg/day, no histopathological or functional disturbances were detected. These results indicate that PQ at doses more than 0.1 mg/kg has toxic effects on the cellular components of the innate immune system of Balb/c mice. The present results, however, indicate that at an exposure level below the recommended acceptable daily intake limit of 0.005 mg/kg, no observable immunotoxicity effect might be expected.

Keywords::

• Paraquat
• phagocytic activity
• immunotoxicity
• CH₅₀

Introduction

The immune system is responsible for protection against infection by pathogens such as bacteria, viruses, and protozoan parasites. These findings therefore suggest how crucial it is to have an intact immune apparatus functioning in harmony with other systems of the body (Bendich, 1993). Conditions that depress immune functions consequently increase the risks of infection and development of certain cancers (Roitt et al., 1985). As the field of immunotoxicology has progressed over the past decades, agrochemical compounds have come to represent important candidates for evaluation of the immunotoxic potentials of widely used chemical compounds (Hassan et al., 2004). It has been reported that certain classes of agents used in agriculture, such as organochlorine, organophosphate and carbamate pesticides, are immuno-toxic (Caroleo et al., 1996). Paraquat (PQ), a non-selective and non-systemic herbicide with a wide spectrum of activity, has been used for several decades in agriculture; however, there is little useful information in the literature regarding its immunotoxicity potential (Schenker et al., 2004). Among what is known, PQ has been shown to induce suppression on the proliferation response of rat splenocytes to Concanavalin A (ConA) (Caroleo et al., 1996; Schenker et al., 2004).

The importance of oxidative stress as a mechanism of PQ toxicity has been demonstrated in studies in plants, bacteria, and in vitro and in vivo systems (Suntres, 2002). It is a fact that this toxic material, capable of entering the body via the food chain, is also harmful to the ecosystem (Dere and Polat, 2001). Although the liver, kidney, heart, and central nervous system are known to be affected, lung damage and pulmonary fibrosis are the most widespread injuries and the usual causes of death following accidental PQ exposures (Mohammadi-Karakani et al., 2006). In our previous study, PQ induced an inhibitory effect upon the cell-mediated and humoral immune responses of Balb/c mice (Riahi et al., 2010).

The acceptable daily intake (ADI) for PQ is 0.005 mg/kg body weight (BW) for humans (FAO, 2003). According to the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) and United States Environmental Protection Agency (US EPA) recommendations, the effects of subacute PQ exposure on some parameters of the immune system (especially innate immunity) were determined at three different doses. The ECETOC and US EPA guidelines are intended to provide information on suppression of the immune system that might occur as a result of repeated exposure to a test chemical. Although some information on potential immunotoxic effects may be obtained from hematology, lymphoid organ weights, and histopathology (in the opinion of ECETOC), there are data from the US EPA that demonstrate that these endpoints alone are not sufficient to predict immunotoxicity (ECETOC, 1994; US EPA, 1998). As such, additional emphasis on effects on the function of cellular and molecular components of the immune system are also needed. Thus, the purpose of the studies reported here was to evaluate innate immune system performance in mice exposed to PQ and to ascertain if the ADI as currently stated might be appropriate after taking into account potential immunomodulatory effects of this pesticide.

Materials and methods

Animals

Male Balb/c inbred mice (6-8-weeks-old; 19–21 g) were purchased from Bu Ali Research Center (Mashhad, Iran). Mice were housed in polystyrene cages in facilities maintained at an ambient temperature of 20–25°C, a relative humidity of 50%, and using a 12-h/12-h light–dark system. All mice were provided access to feed and water ad libitum over the course of the experiment. The mice were acclimatized for 1 week prior to use for exposures/analyses. Thereafter, for the studies herein, the mice were randomly allocated into four treatment groups (at 6 mice/group): three groups were treated with the different doses of PQ and fourth group that was to receive normal saline (NS) vehicle was designated as the negative control. All of the animal experiments here were conducted according to the rules and regulations of the Institutional Animal Ethics Committee, Iran.

Chemicals

PQ dichloride salt and ammonium chloride (NH₄Cl) were purchased from Sigma (Dorset, UK). Fetal bovine serum (FBS) and RPMI-1640 medium were purchased from Gibco (Paisley, UK). Sheep red blood cells (SRBC) were obtained from the Razi Institute (Mashhad, Iran). Sandwich ELISA kits for quantitation of immunoglobulin M (IgM), IgG, and C3 were purchased from Immunology Consultants Laboratory, Inc. (Newberg, OR). The Phagotest kit was purchased from Orpegen Pharma GmbH (Heidelberg, Germany).

Doses and exposure schedules

The mice were divided into four groups (n = 6/group) and were treated with suitable volumes of PQ solutions intraperitoneally (5 ml/kg, prepared in NS) in order to receive 1, 0.1, or 0.01 mg PQ/kg daily for 28 days (5 days/week), respectively. This meant that over the total time of 28 days of the experiment, there were a total of 20 injections. Different mice groups were used for each experiment. Mice in the negative control group received NS during the experimental period.

Determination of hematological parameters

On Day 28, blood was collected from orbital plexuses of the individual animals and total WBC (white blood cell), Hct (hematocrit), and Hb (hemoglobin) levels in each sample were determined. Blood smears were also prepared from each sample and stained with Giemsa to permit differential analyses. All slides were observed under a light microscope; at least 200 cells were counted for each differential analysis.

BW and lymphoid organ weight

On Day 28 (i.e., 2 h after the final dose), mice were sacrificed by cervical dislocation. At post-mortem, the final BW was recorded and the kidneys, liver, spleen, and thymus of each animal were asceptically removed and weighed.

Preparation of single-cell suspension and NKC enumeration

Each spleen isolated above was transferred to a dedicated small petri dish holding 10 ml RPMI-1640 media supplemented with 10% FBS and 2 mM glutamine. For each spleen, the organ was teased between two frosted slides and the tissue dispersion generated was recovered and then centrifuged at 1200 rpm (4°C for 10 min). The supernatant was discarded and the pellet was re-suspended in 3 ml of red blood cell (RBC) lysing buffer containing 0.83% (w/v) NH₄Cl in 100 mM Tris buffer (pH 7.4) and kept at room temperature for 3 min. The cells were then washed three times with the media and suspended into 1 ml of the media containing 10% FBS. Spleen cellularity and viability of cells was performed using the trypan blue exclusion method.

Levels of CD49b cell subtype in the cell population then were determined using a FACSCalibur^™ flow cytometer (BD Biosciences, San Jose, CA) and FITC-conjugated anti mouse pan-natural killer cell (NKC) (FITC rat anti-CD49b with isotype control; eBioscience Inc., San Diego, CA) according to the manufacturer’s protocol. In each case, a minimum of 100,000 events were analyzed per sample. The absolute number in each spleen was determined by multiplying differential ratio of this subtype to the total spleen cell contents (Abuharfeil et al., 2001; Kubosaki et al., 2008).

Histopathological examination

On Day 28, groups of mice were sacrificed by cervical dislocation for use in all histopathological examinations. The thymus, spleen, kidney, lung, and liver of each mouse were then collected and fixed in 10% formalin. Following mounting, 5-µm thick sections of these tissues were stained with Hematoxylin & Eosin (H&E). In addition, the femurs of each mouse were collected and bone marrow smears prepared and stained with H&E. Histopathological changes in these organs were then analyzed via light microscopy and scored based on the degree of changes present (Neishabouri et al., 2004).

Determination of serum levels of complement C3 and immunoglobulin isotypes

To measure the levels of complement C₃ and of IgG and IgM antibodies in the serum samples, commercially available mouse ELISA kits were used (Kubosaki et al., 2008).

CH₅₀ hemolytic assay

Measures of total serum hemolytic activity (CH₅₀) were performed as previously described using rabbit anti-sheep erythrocyte IgG antibodies and sheep erythrocytes (Morgan, 2000). Briefly, a 1:50 dilution of sample sera in gelatin veronal buffer (GVB) (0.1% gelatin, 5 mM veronal, 145 mM NaCl, 0.025% NaN₃ [pH 7.3] buffer) was prepared. This material then underwent a further series of serial dilutions with GVB. Aliquots (50 µl) of each serum dilution were then placed into dedicated tubes. An aliquot (50 µl) of EA (antibody-sensitized sheep erythrocytes, in GVB at 10⁹/ml) preparation was then added. Control tubes for each assay contained 50 µl EA only (to permit assessment of 100% lysis). Cell blanks for each assay contained 50 µl EA and 50 µl GVB (to permit assessment of spontaneous lysis). All tubes were incubated at 37°C for 30 min. Thereafter, to all serum-bearing tubes and cell blanks was added 150 µl ice-cold GVB; to the 100% lysis tubes (control tubes) was added 200 µl H₂O. All tubes were then centrifuged at 1000g (4°C) for 5 min. Aliquots of supernatants (200 µl) from each tube were then transferred to flat-bottom 96-well plates and the absorbance (OD) in each supernatant was measured at 540 nm in a Stat Fax 2100 ELISA plate reader (Awareness Technology, Inc., Palm City, FL). Following correction for background absorbance (i.e., by subtracting cell blank absorbance [value associated with spontaneous lysis] from each value), the fractional hemolysis in each well relative to the 100% lysis wells was calculated:

Fractional hemolytic (y) = (OD serum/OD 100% total lysis)

The amount (in µl) of each serum causing 50% hemolysis (K) as determined by plotting (on log-log graph paper) serum volume in µl added (x) vs. [y/(1− y)] was calculated; this would be expected to yield a linear trace. At 50% hemolysis, y/1− y = 1; hence, the intercept on the x-axis from this point corresponds to 1 CH₅₀ unit for that serum sample.

Phagocytic activity assay

The phagocytic capacity (PC) of monocytes and granulocytes that were present in each sample of mouse blood was measured using a Phagotest kit, and adhering to the manufacturer’s instructions. Each kit contains all necessary reagents for flow cytometric analyses of the phagocytic uptake of fluorescein-labeled opsonized Escherichia coli by the cells. Ingestion of the labeled particles by the phagocytes was evaluated using a BD FACSCalibur^™ flow cytometer. In each case, a minimum of 100,000 events was analyzed per sample. Ultimately, each PC value was expressed as fluorescence intensity/phagocytic cell (Kazlauskaite et al., 2005).

Measurement of general toxicity markers

Activities of serum glutamate oxalate transaminase, aspartate aminotransferase (AST), serum glutamate pyruvate transaminase, alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were estimated by using commercially available kits from Pars Azmoon (Tehran, Iran).

Statistical analysis

Data were analyzed using a Student’s t-test to determine significant differences among the data from the various groups. A p value < 0.05 was considered significant. The values of the data presented are expressed as means ± SE.

Results

Hematological parameters

Both Hb content and Hct levels did not significantly differ between PQ-treated and control (negative) mice. A dose of 1 mg PQ/kg/day was able to cause a significant (p < 0.05) increase in host WBC and neutrophil counts. However, WBC and leukocyte differential levels did not change significantly following the exposures to either lower dose (0.1 or 0.01 mg/kg) of PQ tested (Table 1).

Table 1.  Effects of subacute exposure to paraquat (PQ) IP over 28 days on mice blood markers.

Parameter	1 mg PQ/kg	0.1 mg PQ/kg	0.01 mg PQ/kg	Normal saline (NS)
Hb (g/dl)	13.60 ± 0.25	13.80 ± 0.14	13.90 + 0.11	14.20 ± 0.21
Hct (%)	40.7 ± 0.8	41.2 ± 0.5	41.5 ± 0.5	42.5 ± 0.7
WBC (cell/μl)	10966 ± 428*	8433 ± 458	8583 ± 486	8450 ± 349
Neutrophil	1823 ± 174*	714 ± 91	813 ± 59	653 ± 88
Lymphocyte	8951 ± 550^a	7550 ± 354	7583 ± 496	7700 ± 348
Monocyte	208 ± 44	200 ± 28	210 ± 13	125 ± 17

Data shown are in terms of mean ± SE.

*Value is significantly different (p < 0.05) compared to control group.

^aValue is relatively significant different (p < 0.1) compared to the control group.

BW and lymphoid organ weight

None of the PQ doses tested caused mortality. No significant BW differences were recorded in the various groups of animals either. Furthermore, there was no effect observed with respect to the thymus, liver, and kidney weights at any dose of the PQ. Lastly, the relative thymus, liver, and kidney weights (% BW) did not change significantly with any PQ dose (Table 2). On the other hand, a dose of 1 mg PQ/kg/day was able to significantly (p < 0.05) decrease the weight and relative weight of the spleen when compared values seen in the controls.

Table 2.  Effects of subacute exposure to paraquat (PQ) IP over 28 days on mice organ weights.

Parameter	1 mg PQ/kg	0.1 mg PQ/kg	0.01 mg PQ/kg	Normal saline (NS)
Body weight (g)Before	20.20 ± 0.21	19.90 ± 0.34	20.00 ± 0.24	20.00 ± 0.22
After	19.90 ± 0.22	19.90 ± 0.16	20.20 ± 0.22	20.10 ± 0.18
Spleen weight (mg)	*83.00 ± 1.96	86.33 ± 3.16	91.66 ± 2.66	92.33 ± 2.37
Relative weight % BW	*0.41 ± 0.01	0.43 ± 0.01	0.45 ± 0.01	0.45 ± 0.01
Thymus weight (mg)	62.16 ± 2.30	66.00 ± 2.46	74.50 ± 2.90	67.80 ± 2.13
Relative weight % BW	0.30 ± 0.01	0.32 ± 0.01	0.36 ± 0.02	0.33 ± 0.01
Liver weight (mg)	981 ± 30	973 ± 41	1006 ± 29	966 ± 50
Relative weight % BW	4.91 ± 0.15	4.89 ± 0.23	4.97 ± 0.15	4.78 ± 0.23
Kidney weight (mg)	306.0 ± 6.8	295 ± 7.6	301 ± 12.3	304 ± 6.5
Relative weight % BW	1.53 ± 0.04	1.48 ± 0.04	1.48 ± 0.05	1.50 ± 0.04

Data shown are in terms of mean ± SE.

*Value is significantly different (p < 0.05) compared to control group.

Histopathology

Spleen

Spleen was evaluated for white pulp atrophy (or hyperplasia), red pulp:white pulp ratio, and for the existence of clumps, debris, necrosis, and apoptosis in the white and red pulp regions. In addition, any splenic trabecular disorders were investigated. The analyses revealed that PQ at dose of 1 mg/kg induced splenic white pulp atrophy and an increase in the red:white pulp ratio (minimal observable changes, 1+). These changes were not observed at doses of 0.1 and 0.01 mg/kg/day (Table 3).

Table 3.  Histopathological analysis of mice spleen following subacute exposure to a high dose of PQ IP over 28 days.

Parameter	PQ (1 mg/kg)	Normal saline (NS)
White pulp atrophy	+	−
Capsular-trabecular change	−	−
Red pulp:white pulp ratio	+	−

−: No changes observed, +: minimal observable changes, ++: readily detectable changes.

Thymus

Cortex thickness, relative size of medulla, ratio of cortex to medulla, and capsular changes were evaluated. The thymic tissues were also evaluated for the presence of necrosis, apoptosis, and any abnormal infiltration of cells. Light microscopic examinations of the thymus samples did not reveal any significant effects from the PQ treatments.

Bone marrow

Cellularity, the existence and maturation of hematopoietic cell subtypes, as well as the erythroid:myeloid cell ratio, in each bone marrow specimen isolated was evaluated. Using light microscopic examination, no significant pathologic differences were noted among the samples from the different treatment groups.

Lung

Systematic evaluation related to three compartments of the parenchyma, including the vascular, airways, and alveolus, were separately examined. Light microscopic examinations of the lungs did not reveal any significant differences between the various PQ-treated and control groups.

Liver

Degenerative changes in the liver, cell death (necrosis, apoptosis), inflammatory reaction (acute, chronic, granulomatose), repair changes and fibrosis in different liver zones, ports, and regional centrilobular space were studied. Light microscopic examinations of livers did not show any significant difference between PQ-treated and control groups.

Kidney

Structural changes in the glumeruli, vessels, and tubules were evaluated. Light microscopic examinations of the kidneys did not reveal any significant differences between the PQ-treated and control groups.

Spleen cellularity and NKC levels

PQ at dose of 1 mg/kg/day was able to significantly decrease the cellularity of the spleen when compared with that in the spleens of control mice (p < 0.05). A significant decrease in the absolute levels of CD49b⁺ (NK) cells was seen at dose of 1 mg PQ/kg/day. This is in contrast to the finding that the percentage of CD49b⁺ cells did not significantly differ between PQ-treated mice and the vehicle controls (Table 4).

Table 4.  Effects of subacute exposure to paraquat (PQ) IP over 28 days on mice spleen cellularity and CD49b subtype.

Parameter	1 mg PQ/kg	0.1 mg PQ/kg	0.01 mg PQ/kg	Normal saline (NS)
Spleen cell content (×10^7)	6.44 ± 0.58*	7.98 ± 0.35	8.65 ± 0.33	8.53 ± 0.53
CD49b cell (%)	5.50 ± 0.42	6.40 ± 0.40	5.20 ± 0.87	6.30 ± 0.55
CD49b content (×10^7)	0.35 ± 0.04*	0.51 ± 0.04	0.43 ± 0.06	0.51 ± 0.05

Data shown are in terms of mean ± SE.

^*Value is significantly different (p < 0.05) compared to control group.

Serum IgM, IgG, and complement C3

Measures of serum IgM, IgG, and complement C3 concentrations did not reveal any significant differences between the PQ-treated and negative control groups (Table 5).

Table 5.  Effects of subacute exposure to paraquat (PQ) IP over 28 days on mice humoral components of immune system.

Parameter	1 mg PQ/kg	0.1 mg PQ/kg	0.01 mg PQ/kg	Normal saline (NS)
IgG (μg/ml)	9316 ± 247	9583 ± 283	10033 ± 335	9700 ± 356
IgM (μg/ml)	1021 ± 101	1200 ± 83	1355 ± 144	1228 ± 182
C3 (mg/dl)	30.00 ± 1.00	29.40 ± 0.88	27.00 ± 0.92	27.60 ± 0.82
CH50 (u/μl)	25.6 ± 2.1	29.0 ± 1.8	33.0 ± 2.0	30.7 ± 1.6

Data shown are in terms of mean ± SE.

CH₅₀ hemolytic assay

Analyses of the ability to lyse of target RBCs did not indicate that there were any significant differences between the samples obtained from the PQ-treated and negative control groups (Table 5).

Phagocytic activity assay

PQ at doses of 1 and 0.1 mg/kg/day significantly (p < 0.05) decreased the phagocy-tizing capacity of phagocytic cells (i.e., monocytes and granulocytes). On the other hand, PQ at dose of 0.01 mg/kg/day did not produce any statistically significant changes compared to the negative control group (Figure 1).

Figure 1.  Phagocytosing capacity of blood phagocytic cells of study (given PQ) and control (given NS) mice. *Data are significantly different (p < 0.05) from the control group. Data was expressed in terms of arbitrary units.

General toxicity marker levels

The assessments of serum AST, ALT and LDH activities did not show any significant differences between the PQ-treated and control groups (Table 6).

Table 6.  Effects of subacute exposure to paraquat (PQ) IP over 28 days on general toxicity markers.

Parameter	1 mg PQ/kg	0.1 mg PQ/kg	0.01 mg PQ/kg	Normal saline (NS)
AST (IU/l)	1978 ± 37	2016 ± 55	1933 ± 42	1873 ± 39
ALT (IU/l)	2780 ± 40	2690 ± 37	2656 ± 42	2688 ± 33
LDH (IU/l)	4718 ± 113	4395 ± 76	4426 ± 97	4523 ± 117

Data shown are in terms of mean ± SE.

Discussion

Despite the overt use of PQ as an herbicide in agriculture, there is little information about its influence on the immune system. In a few studies conducted on PQ in rat model, PQ was shown to display clearly immunosuppressive effects (Caroleo et al., 1996). The presence of PQ in the immune and hematopoietic systems, specifically in sites such as the bone marrow, spleen, and thymus, has also been reported (Nagao et al., 1994). As such, it is clear that PQ can enter the immune system of a host at some point during its passage through the body. The biochemical mechanism of PQ toxicity involves its cyclic NADPH cytochrome c-catalyzed reduction and subsequent re-oxidation of the resulting PQ free radical by molecular oxygen (Caroleo et al., 1996). Thus, PQ is a potent producer of superoxide anion (•O₂⁻) and other reactive oxygen species (ROS) in the cells. The results of serum AST, ALT, and LDH analyses showed that the regimen and doses of PQ used in this study did not induce any overt systemic toxicity in the PQ-treated mice.

Our data showed inhibitory effects on macrophages. Doses of 1 and 0.1 mg PQ/kg/day significantly decreased the phagocytizing capacity of monocytes/granulocytes. One mechanism by which the innate branch of the immune system protects a host is by the phagocytic uptake and subsequent killing of pathogens, in part, through an oxidative bactericidal mechanism termed the respiratory burst. The act of ingesting a foreign particle by a macrophage or neutrophil activates NADP oxidase that, in turn, results in the production of a significant amount of •O₂⁻ from molecular oxygen. The •O₂⁻ is then rapidly converted to hydrogen peroxide (H₂O₂) by superoxide dismutase. Neutrophils contain myeloperoxidase that converts H₂O₂ to highly potent bactericidal hypochlorite ions (OCl⁻) (Meydani et al., 1995; Chew and Park, 2004).

Thus, low levels of these substances are essential for daily survival (Boxer et al., 1979; Victor et al., 2004). On the other hand, if it (ROS) is over-produced, or the antioxidant content is low, the cells damage. Phagocytic cells are under oxidative stress when there is an imbalance between pro-oxidants and antioxidants. The intracellular redox (reduction–oxidation) state is important physiologically in terms of maintaining cellular homeostasis (Victor et al., 2004). Phagocytic cells are particularly sensitive to oxidative damage because of the high proportion of polyunsaturated fatty acids in their plasma membranes and their high production of ROS, which contribute to injury. As the concentration of polyunsaturated fatty acids in the membranes is increased, the potential for membrane lipid peroxidation mediated by free radicals also is increased. Lipid peroxidation decreases membrane fluidity, which adversely affects immune responses. Thus, the balance between pro-oxidant production and antioxidant defense is pivotal for a correct cell function, whereas a disturbance in this balance in favour of the oxidants represents an oxidative stress (Hughes, 1999; Victor et al., 2003). Thus, oxidative stress caused by PQ is possibly an important mechanism of PQ-induced immunotoxicity as measured/reflected by decreased phagocyte (i.e., phagocytic) functionality.

A significant decrease in absolute count of CD49b spleen cells was seen at a dose of 1 mg PQ/kg/day. NKC are lymphocytes of the innate immune system that are involved in the early defenses against foreign cells, as well as autologous cells undergoing various forms of stress, such as microbial infection or tumor transformation. There are several reports that oxidative stress can induce apoptosis in natural killer cells (Vivier et al., 2004).

PQ at dose of 1 mg/kg/day was able to significantly increase WBC count as well as neutrophil count (p < 0.05). The increase in WBC and neutrophil counts may be due to a slight inflammation caused by corrosive effects of PQ (at high doses) at the site of injection. PQ at a high dose decreased the spleen cellularity and increased the red pulp:white pulp ratio. In addition, the 1 mg PQ/kg/day dose induced atrophy in the splenic white pulp, suggesting a toxic effect on splenocytes. A significantly increase in red pulp:white pulp ratio may be a reason underlying the relative lymphocytosis, in part due to a suppression of lymphocyte secondary lymphoid tissue homing.

In contrast to the above-noted outcomes, PQ at all doses did not induce any significant effects upon CH₅₀, or upon serum complement C3, total IgM, and total IgG levels. Thus, these results show that PQ has no effect on the synthesis and function of complement system proteins. These outcomes also suggest that B-lymphocyte activity with respect to the production of antibodies is unaffected by a presence of PQ.

In conclusion, this study shows that PQ at a high dose has an inhibitory effect on the phagocytic activity and on the absolute levels of splenic NKC. Our data indicate that PQ at a level of 0.01 mg/kg displays no immunotoxic potential. Therefore, PQ immunotoxicity may not manifest under the ADI level recommended by FAO. More studies are needed to elucidate the exact mechanism by which PQ induced innate immune system toxicity.

Acknowledgements

The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences and Iran National Science Foundation for financial support.

Declaration of interest

The Authors report no conflicts of interest. The Authors are alone responsible for the content and writing of the paper.