Organophosphorus pesticide malathion and immunotoxicity: a more sensitive 1 toxicological target and current recommendations 2

Malathion has been wildly used in agriculture and household pest control for years and can affect human health through drinking water and dietary exposure. This study was to investigate the immunotoxicity of malathion (MLT ） on BALA/c mice for the purpose of providing a scientific basis for pesticides registration management of MLT and other pesticides. based on a 2-year chronic toxicity test and carcinogenicity test in rats, which was used to derive the ADI 39 by Joint WHO/FAO Meeting on Pesticide Residues (0.3 mg/kg bw). It indicates that the immune system 40 is a more sensitive target of MLT toxicity.


Peripheral blood lymphocytes phenotyping 128
Collected whole blood in the same way and transferred into tubes with anticoagulant (EDTA-K 2 ) inside.

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Pathology 146 Animals were humanly sacrificed and dissected at the end of experiment. Liver, kidney, spleen, thymus, 147 lymph glands (axillary lymph nodes, cervical lymph nodes, mesenteric lymph nodes), Peyer's patches 148 and bone marrow were removed. Weighted the organs and calculated the relative weights, and counted 149 the number of Peyer's patches on small intestine. Fixed the organs in 10% neutral buffered formalin, 150 processed for paraffin embedding, sectioned at 5μm, made into slices with HE staining observed under 151 the optical microscope, and all the results were transformed to data through an image analysis system. In 152 addition, the accurate measurements of the splenic periarterial lymphatic sheaths, splenic germinal center, 153 lymph follicles in the Peyer's patches and germinal center in the Peyer's patches were also required.

Plaque-forming cell (PFC) assay 156
Mice had been immunized on day 25 with 0.2 ml of 2% (v/v) SRBC suspensions in sterile saline by 157 intraperitoneal injection. Five days later, the splenocyte suspensions were prepared as stated. Transferred 158 25 μl of splenocyte suspensions in RPMI 1640 medium (5×10 6 cells ml -1 ), which were supplemented 159 with 10% fetal bovine serum and 1% penicillin-streptomycin solution, to a glass tube containing with 50 160 μl of 10% (v/v) SRBC in SA buffer solution and 0.5 ml of agar solution (0.5 g ml -1 in HBSS, pH 7.2-161 7.4), and poured onto slides. The slides were inverted on a special frame after the mixtures were solidified 162 and incubated (37℃, 1.5h). Added guinea pig sera to the slot between the glass slides and the bottom of 163 the frame. Incubated for another 1.5 hour (37℃), then plaque production was counted and the results

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were expressed as the number of PFC/10 6 splenocytes.

Serum immunoglobulin quantification 166
The total IgA, IgG and IgM levels in serum were determined by ELISA kits. The test steps were 167 completely in accordance with the instructions. Mice were immunized as stated. 100 μl of blank, standard 168 or diluted serum samples were respectively added into the 96-well plates in duplicate, incubated (room 169 temperature, 1 h), and washed 4 times with wash solution. Added enzyme-antibody conjugate (100 μl 170 well -1 ), incubated (room temperature, 30 min), and washed 4 times with wash solution. Added TMB 171 substrate solution (100 μl well -1 ), incubated (room temperature, 10 min) and added stop solution (100 μl 172 well -1 ) to prevent further reactions. Finally, determined the absorbance (450 nm) of the contents in each 173 well within 15min, using an ELISA Reader (BioTek., USA.).

Hemolysis test 175
Collected whole blood and got the serum as stated. 1 ml of SA buffer solution, 0.5 ml of 10% (v/v) SRBC, 176 1 ml diluted guinea pig complement (1:8 diluted with SA buffer solution) and 2 μl mouse sera were 177 mixed. Set the control tube without mouse serum. Kept the tubes in a water bath (at 37℃,15 to 30 min) 178 and put them in an ice bath immediately to stop the reaction. Centrifuged (2 000 r min -1 , 10 min) collected

Serum cytokine measurement 186
Collected whole blood without anticoagulant, obtained the serum as stated and assayed for the levels of

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The Carbon-clearance test 242 The activity of macrophage was performed as carbon-clearance test. On day 30, mice were injected with 243 diluted ink (10 mg ml -1 ) via caudal vein. At 2 min or 10 min after the injection, 10 μl of the blood was 244 collected as stated and added into 2 ml of 1 mg ml -1 Na2CO3 solution. Then the absorbance (600 nm) of 245 the mixed samples were measured and the index of phagocytosis was calculated by the formula:

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OD2: the absorbance (600 nm) of 10 min after the injection of ink).

Statistical analysis 250
All data were analysed by using SPSS software (version 17.0). The values were presented as means ± 251 SD. Comparisons between multiple groups were carried out by using one-way ANOVA followed by 14 when equal variances assumption was not met. P<0.05 were considered to statistically significant 254 difference.

Results 256
Effects on body and organ weights 257 As seen in Table 1, there was no significant difference in periodic body weights among each group in all 258 weeks (P>0.05).

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The relative weight of kidney, the absolute and relative weights of liver in the high-dose group were 260 significantly greater than those in the negative control group and other dosage groups (P<0.05); the relative 261 and absolute weights of thymus in the low-dose group and the middle-dose group were significantly higher 262 than those in the negative control group (P<0.05) ( Table 2).

Effects on hematology and clinical chemistry parameters 271
The high-dose of MLT caused a significant decrease in the WBC count, compared with the result of the 272 negative control group (P<0.05), and showed a liner dose-response relationship (Fig. 1).

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Although the PLT count of the high-dose group was significantly lower than that in the low-dose group 274 (P<0.05), there was no significant difference when compared with the negative control group (P>0.05). The

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NEUT level of the low-dose and middle-dose groups were significantly lower than that of the negative control 276 group (P<0.05). Moreover, the count and percentage of BAS in the medium-dose group were higher than 277 that of the negative control group (P<0.05) ( Table 3).

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There were no significant differences in ALT levels between the treated groups and the negative control

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The ALB levels in the high-dose group were significantly lower than those in the negative control group

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Effects on phenotypic analysis of peripheral blood lymphocytes 301 Compared with the negative control group, the percentage of B lymphocytes (CD 3-CD 19+ ) was increased 302 with the dosage of MLT and showed statistical difference in the high-dose group (P<0.05) (Fig.2); the 303 percentage of Ts lymphocytes (CD 3+ CD 8+ ) in the middle-dose group was higher than that of the negative 304 control group (P<0.05), but there was no dose-response relationship (Table 5) .

Effects on the number of splenocytes and marrow cells 311
There was no significant difference in the number of splenocytes (P>0.05) ( Table 6).

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The number of polychromatic normoblast and monocyte series in the medium-dose group were 319 statistically lower than that in the negative control group (P<0.05) ( Table 7).

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Tissues and organs not mentioned above had no visible pathological changes.

PFC and HC50 345
The number of plaque-forming cells was decreased with the rising dose of MLT, and was significantly 346 lower in the high-dose group than that in the negative control group (P<0.05) (Fig. 4). No significant 347 change of HC50 was observed (Table 6).

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Serum immunoglobulin and cytokines 349 23 IgG level in the high-dose group was significantly higher than that in the negative control, the low-dose 350 and the middle dose groups, while there was no dose-response relationship. But the sera IgA level 351 of the high dose group was higher than that in the low-dose and the middle-dose groups (P<0.05). And 352 there was no significant difference in IgM level among the groups (P>0.05) (Table 6, Fig. 5).

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As shown in Table 6, IL-5 level of the middle-dose group was significantly lower than that of the negative 354 control group (P<0.05) ; IL-10 levels in the low-dose and the medium-dose groups were significantly 355 lower than that in the high-dose group, and IL-10 level in the low-dose group was significantly lower than 356 that in the negative control group (P<0.05).

Cellular immunity 358
The mitogen-induced splenic lymphocyte proliferation, CTL cell activity, and DTH reaction were used 359 to detect cellular immunity. Compared with the negative control group, CY inhibited the mitogen-360 induced splenic lymphocyte proliferation and the reaction of DTH. None of these parameters in the high-361 dose group were found to be significantly different (Table 6).

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These results were consistent with the previous studies on the immunosuppressive model, which were 382 induced by CY (21,22). It could be concluded that the immunosuppressed animal model had been 383 successfully established as the positive control in this study.

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The WBC count with differential measured the amount of leukocytes and the percentages of each type,