Antimutagenic and antioxidant activity of novel 4-substituted phenyl-2,2′-bichalcophenes and aza-analogs

Figures & data

Figure 1 4-substituted phenyl-2,2′-bichalcophenes and aza-analogs.

Table 1 Cytotoxicity of bichalcophenes to Salmonella typhimurium TA1535 in liquid medium

Compound^a	Viability at bichalcophene concentrations (% of growth)
	12.5 μM	25 μM	50 μM	100 μM
1A	100.1 ± 10.1	105.5 ± 6.6	107.9 ± 7.6	112.8 ± 11.4
1B	79.1 ± 2.6	59.4 ± 3.7 ^b	46.5 ± 3.4 ^b	40.8 ± 2.5 ^b
2A	139.4 ± 11.2 ^b	124.7 ± 10.1	111.3 ± 10.1	101.2 ± 9.7
2B	123.8 ± 7.7	101.5 ± 9.0	100.4 ± 8.9	94.8 ± 4.8
3A	101.2 ± 6.3	100.0 ± 8.6	100.0 ± 7.3	99.1 ± 8.7
3B	104.4 ± 9.8	108.2 ± 7.4	103.3 ± 5.2	94.7 ± 6.5
4A	134.6 ± 12.4	129.7 ± 9.3	127.1 ± 5.4	116.7 ± 9.6
4B	112.2 ± 5.0	101.3 ± 4.4	92.5 ± 8.0	87.6 ± 8.7
5A	111.1 ± 6.1	110.5 ± 5.6	119.4 ± 6.7	108.5 ± 7.3
5B	92.1 ± 2.3	80.4 ± 3.1	64.5 ± 4.8 ^b	51.4 ± 3.8 ^b

Notes:

a Structures are shown in Figure 1;

b significantly different (P < 0.05) from bacteria grown in the absence of bichalcophenes. The cytotoxicity assays were performed in triplicates and are expressed as % control (mean ± SEM).

Abbreviation: SEM, standard error of the mean.

Table 2 Effect of bichalcophenes on Salmonella typhimurium TA1535 viability as assessed by colony formation on plate

Compound	Viability at bichalcophene concentrations^a (colonies/plate (% of control))
	5/10 μM	10/20 μM
None	45.7 ± 2.2 (100)
1A	54.3 ± 3.3 (119)	45.0 ± 2.5 (98)
1B	61.7 ± 2.7 (135)	55.3 ± 3.6 (121)
2A	88.3 ± 5.4 (193) ^b	47.0 ± 4.5 (103)
2B	44.7 ± 3.6 (98)	46.7 ± 3.2 (102)
3A	46.7 ± 4.8 (102)	42.3 ± 1.9 (93)
3B	42.7 ± 4.8 (93)	43.3 ± 2.5 (95)
4A	41.3 ± 6.4 (90)	63.7 ± 3.8 (139) ^b
4B	39.7 ± 4.5 (87)	40.0 ± 2.1 (88)
5A	40.3 ± 2.9 (88)	45.7 ± 3.0 (100)
5B	41.7 ± 1.8 (91)	41.3 ± 2.8 (90)

Notes:

a Concentrations investigated for all compounds were 10 and 20 μM except for compounds 1B and 5B – the concentrations used were 5 and 10 μM;

b significantly different (P < 0.05) from the number of colonies/plate (mean ± SEM) recorded in the absence of bichalcophenes. Assays were performed in triplicates with a 10⁻⁷ dilution of an overnight Salmonella typhimurium TA1535.

Abbreviation: SEM, standard error of the mean.

Table 3 Effect of bichalcophenes and sodium azide (NaN₃) on Salmonella typhimurium TA1535 viability as assessed by colony formation on plate

Compound	Viability at NaN3 and bichalcophene concentrations^a (colonies/plate (% of control))
	5/10 μM	10/20 μM
None	35.0 ± 3.1 (100)
NaN3 (1 μg/plate)	36.9 ± 4.3 (105)
1A	33.3 ± 3.0 (95)	36.0 ± 1.8 (103)
1B	53.0 ± 1.9 (151) ^b	48.7 ± 4.0 (139)
2A	36.7 ± 3.2 (105)	44.3 ± 3.7 (127)
2B	33.0 ± 2.5 (94)	37.7 ± 2.7 (108)
3A	49.3 ± 2.8 (141)	47.7 ± 3.0 (136)
3B	30.7 ± 2.7 (88)	32.7 ± 3.1 (93)
4A	29.7 ± 2.6 (85)	34.3 ± 2.9 (98)
4B	40.0 ± 3.7 (114)	38.7 ± 3.5 (111)
5A	42.0 ± 4.0 (120)	43.7 ± 3.9 (125)
5B	34.3 ± 3.3 (98)	30.3 ± 1.7 (87)

Notes:

a Concentrations investigated for all compounds were 10 and 20 μM except for compounds 1B and 5B – the concentrations used were 5 and 10 μM;

b significantly different (P < 0.05) from the number of colonies/plate (mean ± SEM) recorded in the absence of bichalcophenes and NaN₃. Assays were performed in triplicates with a 10⁻⁷ dilution of an overnight Salmonella typhimurium TA1535.

Abbreviation: SEM, standard error of the mean.

Table 4 Effect of bichalcophenes, benzo[a]pyrene (B[a]P), and S9 mix on Salmonella typhimurium TA1535 viability as assessed by colony formation on plate

Compound	Viability at B[a]P and bichalcophene concentrations^a (colonies/plate (% of control))
	5/10 μM	10/20 μM
None	63.0 ± 5.9 (100)
B[a]P (20 μM)	67.9 ± 4.7 (108)
1A	62.3 ± 4.1 (99)	64.7 ± 5.1 (103)
1B	63.0 ± 5.9 (100)	64.3 ± 3.7 (102)
2A	60.0 ± 3.8 (95)	61.0 ± 2.1 (97)
2B	65.0 ± 3.9 (103)	62.0 ± 4.8 (98)
3A	64.7 ± 4.0 (103)	65.0 ± 2.9 (103)
3B	60.7 ± 4.4 (96)	59.7 ± 4.7 (95)
4A	64.7 ± 5.7 (103)	64.3 ± 3.0 (102)
4B	67.3 ± 5.0 (107)	61.3 ± 5.0 (97)
5A	65.3 ± 4.5 (104)	66.0 ± 4.8 (105)
5B	62.7 ± 2.9 (100)	59.3 ± 4.0 (94)

Notes:

a Concentrations investigated for all compounds were 10 and 20 μM except for compounds 1B and 5B – the concentrations used were 5 and 10 μM. No significant changes in the viability of Salmonella typhimurium TA1535 treated with bichalcophenes, B[a]P, and S9 mix when compared to non-treated bacteria. Assays were performed in triplicates with a 10⁻⁷ dilution of an overnight bacteria.

Table 5 Determination of mutagenic activity of bichalcophenes in Salmonella typhimurium TA1535 in presence of S9 mix

Compound^a	Bichalcophene mutagenicity (revertant colonies/plate; mean ± SEM)
	5/10 μM	10/20 μM
None (spontaneous)	27.7 ± 2.3
B[a]P (20 μM)	347.7 ± 33.9 ^b
1A	13.3 ± 1.5	23.7 ± 2.5
1B	14.0 ± 2.0	11.3 ± 1.0
2A	29.7 ± 3.2	23.7 ± 2.5
2B	14.3 ± 2.1	16.7 ± 1.9
3A	15.3 ± 4.2	24.7 ± 2.5
3B	11.7 ± 1.5	13.7 ± 1.5
4A	28.7 ± 3.1	34.0 ± 3.0
4B	30.3 ± 3.5	24.0 ± 2.7
5A	15.3 ± 1.5	26.7 ± 2.1
5B	15.7 ± 2.0	13.0 ± 2.0

Notes:

a Concentrations investigated for all compounds were 10 and 20 μM except for compounds 1B and 5B, the concentrations used were 5 and 10 μM;

b significantly different (P < 0.05) from non-treated bacteria (spontaneous mutations). No mutagenic activity of bichalcophenes/S9 mix recorded in Salmonella typhimurium TA1535 when compared to non-treated or B[a]P-treated bacteria. Assays were performed in triplicate.

Abbreviation: SEM, standard error of the mean.

Table 6 Determination of anti-mutagenic activity of bichalcophenes in Salmonella typhimurium TA1535 against sodium azide (NaN₃; 1 μg/plate)

Compound^a	NaN3 mutagenicity at bichalcophene concentrations (revertant colonies/plate; mean ± SEM (% reduction in NaN3 mutagenicity))
	Pre-exposure		Co-exposure
	5/10 μM	10/20 μM	5/10 μM	10/20 μM
NaN3	384.7 ± 14.7 (0)
1A	47.0 ± 3.5 (96) ^b	48.3 ± 6.8 (96) ^b	59.7 ± 9.0 (93) ^b	28.3 ± 3.1 (102)^Table Footnoteb,Table Footnotec
1B	56.7 ± 8.6 (94) ^b	61.7 ± 11.4 (92) ^b	35.0 ± 3.0 (100) ^b	27.3 ± 9.3 (102) ^b
2A	53.0 ± 9.8 (95) ^b	70.7 ± 12.9 (90) ^b	39.3 ± 7.6 (99) ^b	47.3 ± 6.0 (96) ^b
2B	43.7 ± 10.4 (97) ^b	51.0 ± 7.8 (95) ^b	63.0 ± 7.6 (92) ^b	19.3 ± 1.5 (104)^Table Footnoteb,Table Footnotec
3A	47.7 ± 3.5 (96) ^b	52.0 ± 5.0 (95) ^b	95.0 ± 4.0 (83) ^b	42.3 ± 9.1 (98)^Table Footnoteb,Table Footnotec
3B	37.3 ± 2.1 (99) ^b	33.7 ± 3.5 (100)^Table Footnoteb,Table Footnoted	26.7 ± 6.7 (102)^Table Footnoteb,Table Footnoted	17.3 ± 2.1 (105) ^b
4A	186.3 ± 14.2 (57) ^b	103.0 ± 9.2 (80)^Table Footnoteb,Table Footnotec	32.3 ± 4.2 (101) ^b	39.7 ± 6.0 (98) ^b
4B	35.0 ± 4.0 (100)^Table Footnoteb,Table Footnoted	35.0 ± 4.5 (100)^Table Footnoteb,Table Footnoted	80.3 ± 6.1 (87)^Table Footnoteb,Table Footnoted	73.3 ± 7.9 (89)^Table Footnoteb,Table Footnoted
5A	61.3 ± 8.1 (92) ^b	97.7 ± 10.2 (82)^Table Footnoteb,Table Footnotec	47.0 ± 3.5 (96) ^b	32.3 ± 2.5 (101) ^b
5B	35.7 ± 3.1 (100) ^b	37.0 ± 5.3 (99)^Table Footnoteb,Table Footnoted	21.3 ± 6.4 (104)^Table Footnoteb,Table Footnoted	15.3 ± 3.2 (105) ^b

Notes:

a Concentrations investigated for all compounds were 10 and 20 μM except for compounds 1B and 5B – the concentrations used were 5 and 10 μM. Assays were performed in triplicate. The spontaneous revertant colonies were 34.3 ± 4.0;

b significant (P < 0.05) reduction (% of inhibition of mutagenicity indicated in parentheses) from revertant colonies seen with NaN₃, 40% or more reduction means strong antimutagenic activity;

c significant difference (P < 0.05) between bichalcophene concentrations;

d significant difference (P < 0.05) between mononitril (A compounds) versus corresponding monocationic (B compounds).

Abbreviation: SEM, standard error of the mean.

Table 7 Determination of anti-mutagenic activity of bichalcophenes in Salmonella typhimurium TA1535 against benzo[a]pyrene (B[a]P; 20 μM) in presence of S9 mix

Compound^a	B[a]P mutagenicity at bichalcophene concentrations (revertant colonies/plate; mean ± SEM (% reduction in B[a]P mutagenicity))
	Pre-exposure		Co-exposure
	5/10 μM	10/20 μM	5/10 μM	10/20 μM
B[a]P	290.3 ± 21.0 (0)
1A	270.0 ± 25.9 (9)	239.3 ± 22.8 (24) ^b	274.0 ± 25.2 (8)	240.3 ± 27.7 (23)
1B	194.7 ± 16.6 (44) ^b	153.3 ± 5.9 (64)^Table Footnoteb,Table Footnotec	207.3 ± 17.9 (38) ^b	174.3 ± 21.4 (54) ^b
2A	112.7 ± 21.1 (82) ^b	120.0 ± 17.4 (79) ^b	261.0 ± 42.7 (14)	264.0 ± 30.4 (12)
2B	101.7 ± 12.1 (87) ^b	100.0 ± 8.2 (88) ^b	256.3 ± 14.2 (16)	230.7 ± 17.6 (28)
3A	264.0 ± 6.1 (12)	250.0 ± 16.5 (19)	267.0 ± 21.5 (11)	255.7 ± 27.0 (16)
3B	228.0 ± 25.2 (29) ^b	227.7 ± 16.3 (29) ^b	239.7 ± 29.5 (23)	216.3 ± 16.5 (34) ^b
4A	148.7 ± 16.3 (66) ^b	120.3 ± 10.1 (79) ^b	254.7 ± 19.6 (17)	224.7 ± 30.7 (30)
4B	119.7 ± 12.7 (79) ^b	108.0 ± 9.5 (85) ^b	249.7 ± 35.4 (19)	231.7 ± 23.7 (27)
5A	256.7 ± 14.6 (16)	256.0 ± 15.7 (16)	257.7 ± 16.0 (15)	255.7 ± 13.4 (16)
5B	109.0 ± 10.8 (84)^Table Footnoteb,Table Footnotec	95.7 ± 14.1 (90)^Table Footnoteb,Table Footnotec	171.7 ± 27.2 (55)^Table Footnoteb,Table Footnotec	119.3 ± 11.6 (79)^Table Footnoteb,Table Footnotec

Notes:

a Concentrations investigated for all compounds were 10 and 20 μM except for compounds 1B and 5B – the concentrations used were 5 and 10 μM. Assays were performed in triplicate. The spontaneous revertant colonies were 74.7 ± 5.5;

b significant (P < 0.05) reduction (% of inhibition of mutagenicity indicated in parentheses) from revertant colonies seen with B[a]P, 20% or less means no activity, 20%–40% indicates moderate activity, and 40% or more reduction means strong antimutagenic activity;

c significant difference (P < 0.05) between mononitril (A compounds) versus corresponding monocationic (B compounds).

Abbreviation: SEM, standard error of the mean.

Figure 2 The reducing power and total antioxidant activity of bichalcophenes at 25 μM compared to ascorbate at the same concentration.

Table 8 Effects of bichalcophenes on sodium azide (NaN₃) mutant frequency

Compound^#	Mutant frequency and (% of NaN3)^b
	Pre-exposure		Co-exposure
	5/10 μM	10/20 μM	5/10 μM	10/20 μM
NaN3	10.43 (100)
1A	1.41 (14) ^a	1.34 (13) ^a	1.79 (17) ^a	0.79 (8) ^a
1B	1.07 (10) ^a	1.27 (12) ^a	0.66 (6) ^a	0.56 (5) ^a
2A	1.44 (14) ^a	1.60 (15) ^a	1.07 (10) ^a	1.07 (10) ^a
2B	1.32 (13) ^a	1.35 (13) ^a	1.91 (18) ^a	0.51 (5) ^a
3A	0.97 (9) ^a	1.09 (10) ^a	1.93 (18) ^a	0.89 (9) ^a
3B	1.21 (12) ^a	1.03 (10) ^a	0.87 (8) ^a	0.53 (5) ^a
4A	6.27 (60) ^a	3.00 (29) ^a	1.09 (10) ^a	1.16 (11) ^a
4B	0.88 (8) ^a	0.90 (9) ^a	2.01 (19) ^a	1.89 (18) ^a
5A	1.46 (14) ^a	2.24 (21) ^a	1.12 (11) ^a	0.74 (7) ^a
5B	1.04 (10) ^a	1.22 (12) ^a	0.62 (6) ^a	0.50 (5) ^a

Notes:

# Concentrations investigated for all compounds were 10 and 20 μM except for compounds 1B and 5B – the concentrations used were 5 and 10 μM;

a significant (P < 0.05) reduction from mutant frequency seen with NaN₃;

b calculated from mutant colonies (Table 6)/viable colonies (Table 3).

Table 9 Effects of bichalcophenes on benzo[a]pyrene (B[a]P) mutant frequency

Compound^#	Mutant frequency and (% of B[a]P)^b
	Pre-exposure		Co-exposure
	5/10 μM	10/20 μM	5/10 μM	10/20 μM
B[a]P	4.28 (100)
1A	4.33 (101)	3.70 (86)	4.40 (103)	3.71 (87)
1B	3.09 (72) ^a	2.38 (56) ^a	3.29 (77) ^a	2.71 (63) ^a
2A	1.88 (44) ^a	1.97 (46) ^a	4.35 (102)	4.33 (101)
2B	1.56 (37) ^a	1.61 (38) ^a	3.94 (92)	3.72 (87)
3A	4.08 (95)	3.85 (90)	4.13 (96)	3.93 (92)
3B	3.76 (88)	3.81 (89)	3.95 (92)	3.62 (85) ^a
4A	2.30 (54) ^a	1.87 (44) ^a	3.94 (92)	3.49 (82) ^a
4B	1.78 (42) ^a	1.76 (41) ^a	3.71 (87)	3.78 (88)
5A	3.93 (92)	3.88 (91)	3.95 (92)	3.87 (91)
5B	1.74 (41) ^a	1.61 (38) ^a	2.74 (64) ^a	2.01 (47) ^a

Notes:

# Concentrations investigated for all compounds were 10 and 20 μM except for compounds 1B and 5B – the concentrations used were 5 and 10 μM;

a significant (P < 0.05) reduction from mutant frequency seen with B[a]P;

b calculated from mutant colonies (Table 7)/viable colonies (Table 4).