Identification of methylglyoxal as a major mutagen in wood and bamboo pyroligneous acids

Figures & data

Table 1. Mutagenicity tests of pyroligneous acids in Salmonella typhimurium TA100.

Sample	–S9 mix								+S9 mix
	Revertants							Specific mutagenicity (revertants/μL)	Revertants							Specific mutagenicity (revertants/μL)
	Negative control^a	AF-2^b (0.01 μg)	Dose (μL)						Negative control	2AA^c (1.0 μg)	Dose (μL)
			5	10	25	50	100				5	10	25	50	100
W-1	111	411	154	148	275	410	94*	6.0	126	805	152	150	191	281	365*	3.0
W-2	111	411	169	207	597	1522	3426	31.4	126	805	141	149	149	150	209	－
W-3	111	411	126	117	151	222	378	2.5	126	805	129	126	144	146	228	－
W-4	124	605	121	131	175	215	88*	(1.8)^d	123	1196	126	147	156	182	266	1.4
W-5	124	605	132	143	157	207	292	1.7	123	1196	118	131	170	168	166	－
W-6	124	605	122	122	118	131	144	－^e	123	1196	125	125	130	148	150	－
W-7	124	605	128	139	167	218	399	2.5	123	1196	120	124	154	169	175	－
W-8	124	605	120	135	162	169	250	1.2	123	1196	121	130	132	157	163	－
W-9	124	605	136	145	170	287	100*	2.9	123	1196	133	168	166	170	201*	－
W-10	128	734	123	142	219	374	956	7.4	124	984	104	106	125	121	131	－
B-1	111	411	182	227	410	75*	0*	12.0	126	805	142	161	242	260*	90*	3.1
B-2	124	605	121	131	138	143	150	－	123	1196	117	111	108	121	112	－
B-3	111	605	107	106	107	152	325	1.8	126	805	131	133	142	154	200	－
B-4	124	605	126	143	157	178	254	1.3	123	1196	119	120	164	150	201	－
B-5	124	605	137	164	211	269	410	2.9	123	1196	135	160	158	191	251	1.3
B-6	124	605	141	176	221	286	100*	3.4	123	1196	129	140	151	210	170*	－
B-7	124	605	129	153	171	219	409	2.6	123	1196	123	153	161	188	281	1.5
B-8	111	411	109	141	195	466	724	6.1	126	805	140	149	171	216	391	2.4
B-9	124	605	127	147	168	305	84*	3.2	123	1196	119	140	180	198	196*	－
B-10	111	411	132	140	180	375	1031	8.1	126	805	124	121	113	140	169	－

Notes: Numbers of revertants for samples, AF-2 and 2AA are presented as the average of 2 plates, and the number of the negative control revertants is presented as the average of 5 plates. Specific mutagenicity values represent the number of the net revertants per 1 μL of sample, and were calculated from dose–revertant response curves.

^a Distilled water;

^b 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide;

^c 2-Aminoanthracene;

^d Mutagenicity was pseudo-positive;

^e Mutagenicity was negative;

* Growth inhibition was observed.

Table 2. Mutagenicity tests of pyroligneous acids in Salmonella typhimurium TA98.

Sample	–S9 mix								+S9 mix
	Revertants							Specific mutagenicity (revertants/μL)	Revertants							Specific mutagenicity (revertants/μL)
	Negative control^a	AF-2^b	Dose (μL)						Negative control	2AA^c	Dose (μL)
		(0.1 μg)	5	10	25	50	100			(0.5 μg)	5	10	25	50	100
W-1	19	520	21	29	34	47	16*	0.6	26	227	31	31	26	35	35*	−
W-2	19	520	19	24	39	82	37	1.1	26	227	27	30	31	38	31	−
W-3	19	520	24	19	27	44	48	0.3	26	227	29	32	32	40	51	−
W-4	25	501	27	32	29	42	25*	−^d	30	327	32	31	42	46	43*	−
W-5	25	501	26	29	33	35	46	−	30	327	26	33	37	33	36	−
W-6	25	501	26	29	26	33	33	−	30	327	29	31	32	35	37	−
W-7	25	501	30	37	45	53	51	0.6	30	327	32	41	41	40	51	−
W-8	25	501	26	37	38	40	45	−	30	327	31	35	36	38	35	−
W-9	25	501	26	37	36	32	21*	−	30	327	29	49	34	35	31*	−
W-10	25	453	29	22	22	31	45	−	23	245	25	28	26	25	29	−
B-1	19	520	23	33	30	7*	0*	−	26	227	35	32	34	24*	8*	−
B-2	25	501	24	22	22	27	25	−	30	327	26	39	32	34	32	−
B-3	19	520	21	21	28	36	61	0.4	26	227	32	27	33	33	41	−
B-4	25	501	25	32	37	37	47	−	30	327	27	40	33	49	42	−
B-5	25	501	25	38	41	44	53	0.3	30	327	34	38	37	37	48	−
B-6	25	501	29	37	38	53	26*	0.6	30	327	33	42	45	41	28*	−
B-7	25	501	24	29	34	41	54	0.3	30	327	31	34	38	41	40	−
B-8	19	520	24	38	43	102	89*	1.5	26	227	28	30	40	52	60	0.4
B-9	25	501	27	36	33	47	25*	−	30	327	33	39	36	39	20*	−
B-10	19	520	24	22	24	37	58	0.4	26	227	25	30	23	25	37	−

Notes: Numbers of revertants for samples, AF-2 and 2AA are presented as the average of 2 plates, and the number of the negative control revertants is presented as the average of 5 plates. Specific mutagenicity values represent the number of the net revertants per 1 μL of sample, and were calculated from dose–revertant response curves.

^a Distilled water;

^b 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide;

^c 2-Aminoanthracene;

^d Mutagenicity was negative;

* Growth inhibition was observed.

Table 3. Contents of dicarbonyl compounds in pyroligneous acids.

	Content (μmol/mL)			Total (mg/mL)
Sample	G^a	MG^b	DA^c
W-1	5.2	3.7	0.4	0.60
W-2	0.2	0.5	tr^d	0.05
W-3	0.9	1.0	0.1	0.13
W-4	0.1	1.3	0.3	0.13
W-5	0.6	0.6	tr	0.07
W-6	ND^e	ND	ND	−^f
W-7	tr	0.4	ND	0.03
W-8	0.2	0.5	tr	0.05
W-9	0.5	2.4	0.1	0.21
W-10	0.3	0.2	ND	0.03
B-1	5.4	6.5	0.6	0.83
B-2	tr	ND	tr	−
B-3	0.7	0.6	tr	0.08
B-4	0.7	0.5	tr	0.08
B-5	2.3	1.5	0.1	0.25
B-6	0.9	1.0	ND	0.12
B-7	0.4	0.7	0.1	0.08
B-8	1.1	1.2	0.3	0.18
B-9	0.6	2.6	0.2	0.24
B-10	tr	tr	ND	−

Note: Values are the average of 2 times measurement.

^a Glyoxal;

^b Methylglyoxal;

^c Diacetyl;

^d 0.03 μmol/mL ≤ tr < 0.1 μmol/mL;

^e ND < 0.03 μmol/mL;

^f Not calculated.

Fig. 1. HPLC chromatograms of W-1 and B-1 after derivatization.

Notes: Glyoxal, methylglyoxal, and diacetyl in W-1 and B-1 were quantified as quinoxaline (Q), 2-methylquinoxaline (2-MQ), and 2.3-dimethylquinoxaline (2.3-DMQ), respectively, by HPLC after quinoxaline derivatization.

Fig. 2. Mutation incidence rates in methylglyoxal recovery tests in Salmonella typhimurium TA100 in the absence of S9 mix.

Notes: Methylglyoxal (MG) recovery tests were conducted for all mutagenic pyroligneous acids (PA) using the Ames test. PAs were prepared at doses of 20, 40, 60, and 80 µL, and simultaneous Ames tests were performed in PA with and without addition of 0.3 µmol MG; 0.3 µmol MG was used as a reference material. Recovery rates were calculated as percentages after subtracting the number of the net revertants of PA from the number of the net revertants of PA added 0.3 µmol MG and dividing by the number of the net revertants of 0.3 µmol MG. Hence, the recovery rates of MG represent mutation incidence rates of PAs.

Table 4. Mutagenic contribution ratios of dicarbonyl compounds to the mutagenicity of pyroligneous acids in Salmonella typhimurium TA100 in the absence of S9 mix.

	Mutagenic contribution ratio (%)			Total (%)
Sample	G^a	MG^b	DA^c
W-1	8.8	93.0	−^d	101.8
W-2	0.0	1.7	−	1.7
W-3	2.0	52.6	−	54.6
W-4	0.1	31.6	−	31.7
W-5	3.3	54.0	−	57.3
W-6	−	−	−	−
W-7	0.0	16.3	−	16.3
W-8	0.2	17.3	−	17.5
W-9	0.4	70.4	−	70.8
W-10	0.1	1.6	−	1.7
B-1	3.1	78.4	−	81.5
B-2	−	−	−	−
B-3	2.0	32.4	−	34.4
B-4	1.6	24.4	−	26.0
B-5	6.1	61.5	−	67.6
B-6	0.7	17.4	−	18.1
B-7	0.7	28.8	−	29.5
B-8	1.1	28.9	−	30.0
B-9	0.4	84.0	−	84.4
B-10	0.0	0.0	−	0.0

^a Glyoxal;

^b Methylglyoxal;

^c Diacetyl;

^d Not calculated.