. 2022 Jun 17;8(6):e09744. doi: 10.1016/j.heliyon.2022.e09744

Table 7.

Summary of results obtained in the standard (ST) and high bioactivation (HB) crosses of the Drosophila wing SMART after scoring marker-heterozygous flies (mwh +/+ flr³, wild-type wings) treated with nicotine (NIC) at [0.0, 0.0125, 0.0250, 0.050, 0.456, 1.9, 3.8, 11.4 mM]; resveratrol (RES) at [0.0, 0.0004, 0.011 mM]; Milli-Q water and ethanol 1% as dissolvent controls.

Compound Cross^b		Number of flies	Spots per Fly (Number of Spots) Statistical Diagnosis^a					Mean	Clone formation
	Type		Small single spots	large single spots	Twin spots	Total spots	mwhclones	mvh clone size class	per 105 per cell	Cells division^c
			(1-2 cells)	(>2 cells)					observed	control connected
			m = 2	m = 5	m = 5	m = 5
RES [0.0004 mM]
Co-treatments NIC [mM]/RES [0.0004 mM] vs. RES [0.0004 mM]
RES	0.0004 mM	58	0.53 (031)	0.07 (004)	0.00 (0)	0.60 (035)	33	1.64	1.15
NIC	0.456 + RES	33	0.61 (020) -	0.06 (002) -	0.00 (0) -	0.67 (022) -	22	1.45	1.35	0.2
NIC	1.900 + RES	22	0.77 (017) -	0.27 (006) +	0.00 (0) -	1.05 (023) +	23	1.87	2.15	1.0
NIC	3.800 + RES	38	0.42 (016) -	0.05 (002) -	0.00 (0) -	0.47 (018) -	16	1.06	0.85	-0.3
NIC	11.400 + RES	13	0.92 (012) -	0.38 (005) +	0.00 (0) -	1.31 (017) +	17	2.47	2.70	1.5
Co-treatments NIC [mM]/RES [0.0004 mM] vs. NIC
NIC	0.456 mM	38	0.68 (026)	0.05 (002)	0.00 (0)	0.74 (028)	28	1.54	1.50
NIC	0.456 + RES	33	0.61 (020) -	0.06 (002) -	0.00 (0) -	0.67 (022) -	22	1.45	1.35	-0.15
NIC	1.900	46	0.57 (026)	0.04 (002)	0.02 (001)	0.63 (029)	28	1.57	1.25
NIC	1.900 + RES	22	0.77 (017) -	0.27 (006) +	0.00 (0) -	1.05 (023) +	23	1.87	2.25	1.0
NIC	3.800	32	0.62 (020)	0.03 (001)	0.03 (001)	0.69 (022)	22	1.45	1.40
NIC	3.800 + RES	38	0.42 (016) -	0.05 (002) -	0.00 (0) -	0.47 (018) -	16	1.06	0.85	-0.55
NIC	11.400	22	1.27 (028)	0.23 (005)	0.05 (001)	1.55 (034)	34	1.71	3.15
NIC	11.400 + RES	13	0.92 (012) -	0.38 (005) +	0.00 (0) -	1.31 (017) +	17	2.47	2.65	-0.5
RES [0.011 mM]
Co-treatments NIC [mM]/RES [0.011 mM] vs. RES [0.011 mM]
RES	0.011 mM	58	0.47 (027)	0.03 (002)	0.07 (004)	0.57 (033)	32	1.84	1.15
NIC	0.456 + RES	45	0.69 (031) -	0.04 (002) -	0.07 (003) -	0.80 (036) -	36	1.86	1.65	0.5
NIC	1.900 + RES	42	0.76 (032) +	0.10 (004) -	0.12 (005) -	0.98 (041) +	41	2.02	2.00	0.85
NIC	3.800 + RES	52	0.62 (032) -	0.12 (006) -	0.04 (002) -	0.77 (040) -	40	1.77	1.60	0.45
NIC	11.400 + RES	37	0.38 (014) -	0.14 (005) +	0.05 (002) -	0.57 (021) -	20	2.30	1.15	0.00
o-treatments NIC [mM]/RES [0.011 mM] vs. NIC
NIC	0.456 mM	38	0.68 (026)	0.05 (002)	0.00 (0)	0.74 (028)	28	1.54	1.5
NIC	0.456 + RES	45	0.69 (031) -	0.04 (002) -	0.07 (003) -	0.80 (036) -	36	1.86	1.65	0.15
NIC	1.900	46	0.57 (026)	0.04 (002)	0.02 (001)	0.63 (029)	28	1.57	1.25
NIC	1.900 + RES	42	0.76 (032) -	0.10 (004) -	0.12 (005) +	0.98 (041) +	41	2.02	2.00	0.75
NIC	3.800	32	0.62 (020)	0.03 (001)	0.03 (001)	0.69 (022)	28	1.45	1.40
NIC	3.800 + RES	52	0.62 (032) -	0.12 (006) -	0.04 (002) -	0.77 (040) -	40	1.77	1.55	0.15
NIC	11.400	22	1.27 (028)	0.23 (005)	0.05 (001)	1.55 (034)	34	1.71	3.15
NIC	11.400 + RES	37	0.38 (014) ↓	0.14 (005) -	0.05 (002) -	0.57 (021) ↓	20	2.30	1.10	-2.05
Co-treatments NIC [mM]/RES [0.011 mM] vs. RES [0.011 mM]
RES	0.011 mM	99	0.58 (057)	0.05 (005)	0.04 (004)	0.67 (066)	65	1.58	1.35
NIC	0.0125 + RES	40	0.73 (029) -	0.00 (000) -	0.00 (000) -	0.73 (029) -	29	1.14	1.50	0.15
NIC	0.0250 + RES	49	0.96 (047) +	0.27 (013) -	0.00 (000) -	1.22 (060) +	59	2.08	2.45	1.10
NIC	0.0500 + RES	53	0.62 (033) -	0.04 (002) -	0.00 (000) -	0.66 (035) -	35	1.34	1.35	0.00
Co-treatments NIC [mM]/RES [0.011 mM] vs. NIC
NIC	0.0125	44	1.16 (051)	0.43 (019)	0.00 (000)	1.59 (070)	70	2.26	3.25
NIC	0.0125 + RES	40	0.73 (029) ↓	0.00 (000) ↓	0.00 (000) -	0.73 (029) ↓	29	1.14	1.50	-1.8
	0.0250	41	0.66 (027)	0.00 (000)	0.02 (001)	0.68 (028)	28	1.21	1.40
NIC	0.0250 + RES	49	0.96 (047) -	0.27 (013) -	0.00 (000) -	1.22 (060) +	59	2.08	2.45	1.05
NIC	0.050	21	0.67 (014)	0.10 (002)	0.05 (001)	0.81 (017)	17	1.47	1.66
NIC	0.050 + RES	53	0.62 (033) -	0.04 (002) -	0.00 (000) -	0.66 (035) -	35	1.34	1.35	-0.31
HB
RES 0.0004 mM
RES	0.0004	87	0.52 (045)	0.06 (005)	0.01 (001)	0.59 (051)	51	1.84	1.20
NIC	0.456 + RES	64	0.36 (023) -	0.11 (007) -	0.00 (000) -	0.47 (030) -	29	2.21	0.95	-0.25
NIC	1.900 + RES	49	0.29 (014) ↓	0.08 (004) -	0.02 (001) -	0.39 (019) -	18	2.33	0.75	-0.45
NIC	3.800 + RES	78	0.59 (046) -	0.05 (004) -	0.03 (002) -	0.67 (052) -	52	1.63	1.35	0.15
NIC	11.400 + RES	10	0.40 (004) -	0.00 (000) -	0.00 (000) -	0.40 (004) -	04	1.25	0.80	-0.40
NIC	0.456	99	0.38 (038)	0.07 (007)	0.03 (003)	0.48(048)	47	1.64	0.95
NIC	0.456 + RES	64	0.36 (023) -	0.11 (007) -	0.00 (000) -	0.47 (030) -	29	2.21	0.90	-0.05
NIC	1.900	58	0.47 (027)	0.05 (003)	0.07 (004)	0.59 (034)	34	1.82	1.20
NIC	1.900 + RES	49	0.29 (014) -	0.08 (004) -	0.02 (001) -	0.39 (019) -	18	2.33	0.75	-0.45
NIC	3.800	69	0.36 (025)	0.06 (004)	0.00 (000)	0.42 (029)	28	1.61	0.85
NIC	3.800 + RES	78	0.59 (046) +	0.05 (004) -	0.03 (002) -	0.67 (052) +	52	1.63	1.40	0.55
RES 0.011 mM
RES	0.011	59	0.36 (021)	0.02 (001)	0.02 (001)	0.39 (023)	23	1.43	0.80
NIC	0.456 + RES	60	0.37 (022) -	0.10 (006) -	0.02 (001) -	0.48 (029) -	29	1.97	1.00	0.20
NIC	1.900 + RES	60	0.34 (021) -	0.10 (006) -	0.05 (003) -	0.49 (030) -	29	2.03	1.00	0.20
NIC	3.800 + RES	60	0.45 (027) -	0.22 (013) +	0.00 (000) -	0.67 (040) -	40	2.42	1.35	0.55
Co-treatments NIC [mM]/RES [0.011 mM] vs. NIC
NIC	0.456	60	0.33 (020)	0.08 (005)	0.02 (001)	0.43 (026)	25	1.64	0.85
NIC	0.456 + RES	60	0.37 (022) -	0.10 (006) -	0.02 (001) -	0.48 (029) -	29	1.97	1.00	0.15
NIC	1.900	58	0.47 (027)	0.05 (003)	0.07 (004)	0.59 (034)	34	1.82	1.2
NIC	1.900 + RES	60	0.34 (021) -	0.10 (006) -	0.05 (003) -	0.49 (030) -	29	2.03	1.0	0.20
NIC	1.900 + RES	60	0.34 (021) -	0.10 (006) -	0.05 (003) -	0.49 (030) -	29	2.03	1.0	0.20
NIC	1.900 + RES	60	0.34 (021) -	0.10 (006) -	0.05 (003) -	0.49 (030) -	29	2.03	1.0	0.20
NIC	3.800 + RES	60	0.45 (027) -	0.22 (013) +	0.00 (000) -	0.67 (040) +	40	2.42	1.35	0.55
Co-treatments NIC [mM]/RES [0.011 mM] vs. RES [0.011 mM]
RES	0.011 mM	60	0.73 (044)	0.07 (004)	0.02 (001)	0.82 (049)	48	1.56	1.65
NIC	0.0125 + RES	67	0.54 (036) -	0.00 (000) -	0.04 (003)-	0.58 (039) -	39	1.54	1.20	-0.45
NIC	0.0250 + RES	59	0.68 (040) -	0.07 (004) -	0.00 (000) -	0.75 (044) -	44	1.36	1.55	-0.10
NIC	0.0500 + RES	57	0.63 (036) -	0.05 (003) -	0.00 (000) -	0.68 (039) -	39	1.33	1.40	-0.25
Co-treatments NIC [mM]/RES [0.011 mM] vs. NIC
NIC	0.0125	60	0.68 (041)	0.05 (003)	0.02 (001)	0.75 (045)	42	1.14	1.45
NIC	0.0125 + RES	67	0.54 (036) -	0.00 (000) -	0.04 (003)-	0.58 (039) -	39	1.54	1.2	-0.25
NIC	0.0250	60	0.65 (039)	0.03 (002)	0.00 (000)	0.68 (041) -	41	1.29	1.4
NIC	0.0250 + RES	59	0.68 (040) -	0.07 (004) -	0.00 (000) -	0.75 (044) -	44	1.36	1.55	0.15
NIC	0.050	60	0.77 (046)	0.08 (005)	0.00 (000)	0.85 (051) -	51	1.61	1.75
NIC	0.050 + RES	57	0.63 (036) -	0.05 (003) -	0.00 (000) -	0.68 (039) -	39	1.33	1.4	-0.35

Statistical diagnoses according to Frei and Würgler (1988) m: minimal risk multiplication factor for the assessment of negative results. For the final statistical diagnosis of all positive (+) and negative (-) results; (↓) significant decrease; the non-parametric Mann-Whitney and Wilcoxon U-test with significance levels α and β = 0.05 was used to exclude false positive or negative diagnoses [Frei and Würgler, 1995]. One side binomial test, significance levels α and β: significative results: + (α ≤ 0.05; no significant results: - (β ≤ 0.05).

ST: standard cross; HB: high bioactivation cross.

Clone frequencies per fly divided by the number of cells examined per fly (48,800) gives an estimate of formation frequencies per cell and per cell division in chronic exposure experiments [Frei and Würgler, 1995].