Mechanism of perfluorooctanesulfonate (PFOS)-induced apoptosis in the immunocyte

Figures & data

Table 1.  Effects on serum corticosterone, body weight, and organ indices in mice treated orally with PFOS daily for 7 days.

PFOS (mg/kg/day)	n	Serum PFOS (mg/L)	Serum corticosterone (ng/ml)	Body weight change^a	Food intake in 7^th day	Relative splenic weight^b	Relative thymic weight^b	Relative kidney weight^b	Relative hepatic weight^b
Control	12	0.04 ± 0.01	137.76 ± 19.32	1.13 ± 0.15	5.97 ± 0.28	0.45 ± 0.03	0.22 ± 0.03	1.34 ± 0.03	5.27 ± 0.05
1	12	24.37 ± 1.13*	141.25 ± 18.76	1.18 ± 0.22	6.12 ± 0.35	0.47 ± 0.05	0.27 ± 0.04	1.41 ± 0.04	5.66 ± 0.07
5	12	87.56 ± 5.49*	156.94 ± 20.15	1.01 ± 0.24	5.48 ± 0.26	0.42 ± 0.02	0.19 ± 0.02	1.23 ± 0.03	6.48 ± 0.14*
10	12	126.49 ± 12.75*	197.83 ± 24.39	0.49 ± 0.11	4.23 ± 0.31*	0.33 ± 0.04	0.14 ± 0.02	1.36 ± 0.03	8.03 ± 0.22*

*Value significantly different from respective control at p ≤ 0.05. Data reported as mean ± SE. Body mass and organ mass data did not require transformation for statistical analysis.

^a Body weight change denotes change in body weight (in grams) from start to finish: [Final body weight (g) – start body weight (g)].

^b Calculated as: [organ weight (g)/body weight (g)] × 100.

Table 2.  Distribution apoptotic and necrotic cells in spleens from mice treated daily with PFOS orally for 7 days.

PFOS (mg/kg/day)	Cell cycle study (% of sub-G1 population)		Annexin binding assay
			Splenocytes		Thymocytes
	Splenocytes	Thymocytes	Apoptotic	Necrotic	Apoptotic	Necrotic
Control	4.29 ± 0.37	3.27 ± 0.28	3.54 ± 0.21	1.02 ± 0.15	2.68 ± 0.22	0.79 ± 0.08
1	5.88 ± 0.42	3.96 ± 0.33	4.17 ± 0.23	0.85 ± 0.12	3.49 ± 0.35	0.95 ± 0.11
5	7.46 ± 0.63*	4.82 ± 0.46	10.59 ± 0.65*	1.33 ± 0.14	7.81 ± 0.54*	1.46 ± 0.27
10	10.35 ± 0.81*	7.79 ± 0.65*	16.82 ± 0.71*	1.16 ± 0.16	12.56 ± 0.79*	2.38 ± 0.35*

*Value significantly different from respective control at p ≤ 0.05. Data reported as mean ± SE. n = 12/group. Values for the Annexin assay are in terms of percentage of all cells counted.

Figure 1.  Effects of PFOS on DNA fragmentation (mono- and oligonuclesomes). Thymocytes and splenocytes were isolated from male C57Bl/6 mice following oral exposures to PFOS for 7 days. DNA fragmentation was determined by Cell Death Detection ELISA. Data are presented as mean (± SE); n = 12/group. When significant differences were detected by the F-test (p < 0.05), a Dunnett’s t-test was used to compare treatment groups to the control group. *Value significantly different from control at p ≤0.05.

Figure 2.  Effects of PFOS on mitochondrial membrane potential. Thymocytes and splenocytes were isolated from male C57Bl/6 mice following oral exposures to PFOS for 7 days. Rh-123 was added and the cells were incubated for 60 min. Fluorescence was measured using a flow cytometer with an FL-1 filter. Data are presented as mean (± SE); n = 12/group. When significant differences were detected by the F-test (p < 0.05), a Dunnett’s t-test was used to compare treatment groups to the control group. *Value significantly different from control at p ≤ 0.05.

Figure 3.  Effects of PFOS on generation of ROS. Thymocytes and splenocytes were isolated from male C57Bl/6 mice following oral exposures to PFOS for 7 days. DCFH-DA was added and the cells were incubated for 60 min. DCF fluorescence was measured using a flow cytometer with an FL-1 filter. Data are presented as mean (± SE); n = 12/group. When significant differences were detected by the F-test (p < 0.05), a Dunnett’s t-test was used to compare treatment groups to the control group. *Value significantly different from control at p ≤ 0.05.

Table 3.  Anti-oxidative enzyme activity and GSH content in splenocytes from mice treated daily with PFOS orally for 7 days.

PFOS (mg/kg/day)	GSH	SOD	CAT	GR	GST	GPx
Control	178.93 ± 15.28	62.10 ± 7.54	16.33 ± 1.71	18.21 ± 2.03	149.90 ± 13.82	72.35 ± 5.46
1	166.49 ± 16.40	57.89 ± 6.38	17.96 ± 2.28	20.15 ± 2.16	154.76 ± 14.39	64.19 ± 6.78
5	127.55 ± 14.72*	88.91 ± 5.17*	25.48 ± 3.15	29.73 ± 3.41*	111.38 ± 12.66	51.09 ± 5.31*
10	91.36 ± 11.63*	105.63 ± 10.25*	31.57 ± 3.69*	34.46 ± 3.72*	89.72 ± 7.57*	42.86 ± 5.22*

*Value significantly different from respective control at p ≤ 0.05. Data reported as mean ± SE.

GSH, Glutathione; SOD, Superoxide Dismutase; CAT, Catalase; GR, Glutathione Reductase; GST, Glutathione-S-Transferase; GPx, Glutathione Peroxidase. SOD, CAT, GST and GPx activities were expressed as U/mg protein, GR activity as U/g protein, and GSH content as mg GSH/g protein. Data did not require transformation for statistical analysis. n = 12/group.

Table 4.  Expression of bcl-2, p53, bax, caspase-3, and caspase-9 proteins in splenic lymphocytes from mice treated daily with PFOS orally for 7 days.

PFOS (mg/kg/day)	Bcl-2	p53	Bax	Caspase-3	Caspase-9
Control	35.22 ± 3.45	21.67 ± 2.49	24.11 ± 3.05	17.54 ± 2.06	22.38 ± 2.80
1	37.84 ± 3.72	26.13 ± 2.33	27.83 ± 2.97	19.33 ± 2.41	23.37 ± 2.15
5	24.37 ± 2.38*	39.51 ± 3.74*	30.62 ± 3.36	28.62 ± 2.15*	28.64 ± 3.12
10	20.22 ± 2.59*	44.29 ± 3.62*	35.78 ± 3.93*	32.04 ± 3.23*	33.79 ± 3.44*

*Value significantly different from respective control at p ≤ 0.05. Data reported as mean ± SE.

Mice splenic lymphocytes were incubated with anti-bcl-2/-p53/-bax/-caspase-3/-caspase-9 antibodies and then with FITC-conjugated second antibodies. The cells were analyzed on a flow cytometer. Data did not require transformation for statistical analysis. n = 12 /group.

Values shown are in terms of percentage of all cells counted.