The α₂M protein was isolated and purified from human blood plasma using ammonium sulfate precipitation followed by gel exclusion chromatography as per the method described previously3. A 5% (w/v) native PAGE was performed21 and the gel was stained with Coomassie brilliant blue R-250 (0.15% in 10% acetic acid). The gel was de-stained for 12 h in the de-staining solution (10% acetic acid), and the purified α₂M formed a single band on the gel.

The SH-SY5Y cell line was cultured in a medium containing 1:1 DMEM and Ham’s F12 medium, 10% FBS, and 1% penicillin-streptomycin26. The cells were treated with a standard solution of pesticides and α₂M, accordingly, to perform the experiments. Cells were used at 3–7 passages. The cells were divided into five groups based on the treatment with pesticides and proteins to obtain results for various stress markers. Group I was the control group comprising only SH-SY5Y cells under standard conditions (37 °C and 5% CO₂). Group II consisted of SH-SY5Y cells incubated with α₂M. The concentration of protein was 2 μM and the incubation time was 3 h (37 °C). Group III comprised pesticide (CPF, ALD, and DLM)-treated SH-SY5Y cells. Cells were treated with 5 μM of each pesticide (CPF, ALD, and DLM) separately for 3 h under standard conditions (37 °C and 5% CO₂). Group IV was the pesticides (CPF, ALD, and DLM) and α₂M group, in which the SH-SY5Y cells were treated with 5 μM of the pesticides for 3 h and then treated with α₂M for 3 h.

To determine the cytotoxicity and cell viability of the SH-SY5Y cells, the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide MTT assay was performed in 96-well plates27. The cells were used at 70 – 80% confluence. Later, the medium was removed and the cells were treated with pesticides (CPF, ALD, and DLM; 5 μM) for 6 h and later incubated with α₂M for 3 h. Afterward, the cells were treated with MTT solution (5 mg/mL stock solution) at a final concentration of 0.5 mg/mL MTT. The cells were incubated for 4 h at 37 °C in 5% CO₂. Finally, the cells were treated with 100 μL DMSO for 10 min to dissolve the formazan crystals. The absorbance was measured using an ELISA plate reader at 570 nm with a reference wavelength of 630 nm and was directly proportional to the number of viable cells. The experiments were performed in triplicate.

ROS production was measured using DCFH-DA28. The cells were treated with α₂M and the pesticides, following which 10 μM DCFH-DA was added to the medium for 1 h at 37 °C for diffusion into the cells. A multi-detection microplate reader was used for fluorescence measurement. The excitation and emission wavelengths for DCFH-DA were 485 and 535 nm, respectively28, 29, 30.

To detect the generation of ROS by pesticides in the neuronal cell line, the MDA levels were measured. The MDA levels in SH-SY5Y cells were quantified with the TBA reaction. Thiobarbituric acid reactive substances (TBARS) were measured by comparing the absorption to the standard curve of MDA equivalents generated by the acid-catalyzed hydrolysis of tetramethoxypropane31. The absorbance was recorded at 532 nm.

The antioxidant enzyme SOD converts superoxide (O₂^-.) into H₂O₂ and O₂, which is converted to water by other enzymes. The measurement was carried out as described previously32. To 80 μL of the cell suspension, 2.82 ml of 0.05 mM tris-succinate buffer was added, and the sample was treated for 30 min in a CO₂ incubator. The reaction was initiated by adding 100 μL of 8 mM pyrogallol solution to each well32. The absorbance was read at 420 nm.

GPx are cytosolic enzymes that catalyze the conversion of H₂O₂ into H₂O and O₂ and the reduction of peroxide radicals (ROO^.) into alcohol and oxygen. GPx levels were measured by determining the decrease in absorbance at 340 nm upon the oxidation of NADPH to NADP⁺ 33.

All experiments were repeated thrice and the data shown is the mean +- SD. P-value < 0.05 is considered as significant difference.

Figure 2

Figure 3

Figure 4

Table 1

Enzyme	Control	CPF	ALD	DLM
SOD	1.6 ± 0.140	0.90 ± 0.086	1.4 ± 0.131	0.88 ± 0.041
GPx	24.4 ± 2.25	18.3 ± 1.31	20 ± 0.100	16.3 ± 0.07

SOD and GPx measurements are reported in μmol/min/mg cells for SOD and nmol/min/mg cells for GPx (SOD: superoxide dismutase; GPx: glutathione peroxidase).

Figure 5

The MTT assay was performed to assess cell viability and cell proliferation activity, in which the quantity of formazan is directly proportional to the number of viable cells. The SH-SY5Y cells treated with all three pesticides (CPF, ALD, and DLM) in group III showed reduced cell viability (p < 0.001; Figure 1). Almost no effect on viability was seen for cells treated with α₂M (group II), while the effect of the pesticides appeared to be reversed in cells treated with pesticides after incubation with α₂M (group IV).

Figure 3 shows the ROS levels induced by the pesticides as monitored by DCFHDA in the control group of SH-SY5Y cells and all three pesticides in group III (p < 0.001). The increased fluorescence in group III as compared to the control group and α₂M group indicates the production of ROS. On the contrary, group IV showed a reduction in the fluorescence intensity as compared to group III, indicating the defensive function of α₂M against ROS production.

Figure 4 shows the TBARS activity in the SH-SY5Y cells in various groups. The highest MDA production was recorded in group III, in which the cells were treated with pesticides. However, group II showed no TBARS production. Group IV (pesticides + cells + α₂M), however, showed a reduction in the TBARS level as compared to group III. The MDA levels were normal in the control group and slightly reduced in the α₂M group (p < 0.001). However, MDA levels were significantly decreased in the α₂M + pesticides + cells group (p < 0.001) compared to the pesticides group. Hence, the protective effects of α₂M were observed with respect to MDA levels due to pesticide-triggered ROS in SH-SY5Y cells.

Figure 5 shows the effect of various treatments on SOD levels in the control and other groups. Group III (pesticides group) showed reduced SOD activity as compared to group I (control) and group II (α₂M; p < 0.001), demonstrating the toxicity triggered by the pesticides. On the other hand, group IV (α₂M + pesticides + SH-SY5Y cells) showed a remarkable increase in SOD and GPx activity levels, indicating the protective effect of α₂M against the pesticides.Table 1 shows the effect of pesticides on the activity of antioxidant enzymes in the cells. The pesticides group showed reduced GPx activity as compared to the control group I and α₂M group II, while group IV (α₂M and pesticides) showed increased GPx activity levels.