Acrylonitrile’s genotoxicity profile: mutagenicity in search of an underlying molecular mechanism

Figures & data

Figure 1. Metabolism of acrylonitrile. *Reaction can also occur nonenzymatically; P450: Cytochrome P450; GST: Glutathione-S-Transferase; GSH: Reduced glutathione; EH: Epoxide Hydrolase; RH: Rhodanese; Px: Peroxidase; XO: Xanthine oxidase; CHEMA: N-acetyl-S-(1-cyano-2-hydroxyethyl)-L-cysteine; CEMA: N-acetyl-S-(2-cyanoethyl)-L-cysteine; ATCA: 2-aminothiazoline-4-carboxylic acid; CN-: cyanide ion; SCN-: thiocyanate OSCN-: hypothiocyanite.

Figure 2. Ratio of ACN urinary metabolites CHEMA:CEMA in humans using NHANES (2015–2016,) data. “X”: data point for individuals with both metabolites detectable in urine. Solid line: linear regression.

Figure 3. Chemically induced genotoxicity: a continuum that may produce mutation (Albertini and Kaden 2020).

Table 1. ACN/CNEO induced generic DNA damage.

Endpoint	Methods	Results, comments	Reference
Chemical reactivity (in vitro)	1 mM ACN, 100 mM CNEO in calf thymus DNA (Guengerich et al. 1981) ACN in isolated DNA (Peter, Appel, et al. 1983) Up to 100 µM AN in DNA from TK6 cells (Walker, Walker, et al. 2020)	Covalent binding to isolated DNA or nucleosides	Guengerich et al. 1981; Peter, Appel, et al. 1983; Walker, Walker, et al. 2020
Chemical reactivity (in vivo)	0.6 mg/kg CNEO ip (Hogy and Guengerich 1986) 0.2 mM ACN ip (Peter, Appel, et al. 1983) 46.5 mg/kg ACN oral (Farooqui and Ahmed 1983; Ahmed, Abdel-Aziz, et al. 1992; Ahmed, Abdel-Rahman, et al. 1992; Abdel-Rahman et al. 1994) 11 mg/kg ACN oral (Williams et al. 2017)	Covalent binding to DNA in rodents; confounded by protein binding	Hogy and Guengerich 1986; Peter, Appel, et al. 1983; Farooqui and Ahmed 1983; Ahmed, Abdel-Aziz, et al. 1992; Ahmed, Abdel-Rahman, et al. 1992; Abdel-Rahman et al. 1994; Williams et al. 2017
DNA damage; SS-breaks/apurinic sites (in vitro)	200–400 µg/ml ACN in SHE cells (Parent and Casto 1979) 1 mM CNEO in SV40 phage DNA (Peter, Schwarz, et al. 1983) 65.8 µg/ml ACN in rat hepatocytes (Bradley 1985) 0.07–0.1 M ACN in CHO cells (Douglas et al. 1985) 200–500 µg/ml ACN in human bronchial epithelial cells (Chang et al. 1990) 50 mM CNEO in plasmid DNA (Yates et al. 1994) 0.32–5.6 mM ACN in rat and human hepatocytes (Robbiano et al. 1994)	SS-breaks in cultured cells or isolated DNA in vitro by alkaline elution or fluorescence screening	Parent and Casto 1979; Peter, Schwarz, et al. 1983; Bradley 1985; Douglas et al. 1985; Chang et al. 1990; Yates et al. 1994; Robbiano et al. 1994
	Unspecified concentration of ACN in calf thymus DNA (Kailasam and Rogers 2007) 1 mM ACN in rat and human astrocytes (Pu et al. 2006) 0.1–1 mM ACN in rat astrocytes (Klaunig and Forney 2010)	Failure to find breaks in cultured cells or isolated DNA in vitro by fluorescence screening or standard comet assays at concentrations up to 1 mM	Kailasam and Rogers 2007; Pu et al. 2006; Klaunig and Forney 2010
DNA damage; SS-breaks/apurinic sites (in vivo)	Unspecified dose of ACN ip in rats (Hachiya et al. 1984, 1986) 30 mg/kg ACN ip in rats (Sekihashi et al. 2002) 13.7–62.5 mg/kg ACN gavage in rats (Nakagawa et al. 2015) 10–60 mg/kg ACN gavage in mice (Walker, Walker, et al. 2020)	SS breaks in cells in vivo in rodents by alkaline elution or standard alkaline comet assays at doses from 15.7 mg/kg to 0.5 LD50	Hachiya et al. 1984, 1986; Sekihashi et al. 2002; Nakagawa et al. 2015; Walker, Walker, et al. 2020
	100–200 ppm ACN in dw in rats (Pu et al. 2009, 2015; Williams et al. 2017)	Failure to find SS breaks in cells in vivo in rodents by standard alkaline comet assays at doses from 3 to 300 ppm in drinking water	Pu et al. 2009, 2015; Williams et al. 2017
DNA repair: UDS liquid scintillation counting (in vitro)	0.5 M ACN in human lymphocytes (Perocco et al. 1982) up to 0.036 mM ACN in HeLa cells (Rizzi et al. 1984) 0.5 µg/ml ACN in rat primary hepatocytes (Glauert et al. 1985)	Three studies reported positive UDS responses in cultured cells or human PBLs over an ACN dose range of 0.05–530 µg/ml	Perocco et al. 1982; Rizzi et al. 1984; Glauert et al. 1985
	2.5 mg/ml ACN in HeLa cells	A single study in HeLa cells was negative	Martin and Campbell 1985
DNA repair: UDS liquid scintillation counting (in vivo)	50 mg/kg ACN by gavage in rats (Hogy and Guengerich 1986) 46.5 mg/kg ACN gavage in rats (Ahmed, Abdel-Aziz, et al. 1992; Ahmed, Abdel-Rahman, et al. 1992; Ahmed et al. 1996; Abdel-Rahman et al. 1994)	Several studies reported positive UDS responses in ACN exposed rats at oral doses up to 50 mg/kg	Hogy and Guengerich 1986; Ahmed, Abdel-Aziz, et al. 1992; Ahmed, Abdel-Rahman, et al. 1992; Abdel-Rahman et al. 1994; Ahmed et al. 1996
DNA Repair: UDS Autoradiography (in vitro)	0.026–53 µg/ml ACN in rat hepatocytes (Probst and Hill 1985) 0.01–1 mM AN; 0.01–0.1 mM CNEO in rat hepatocytes (Butterworth et al. 1992) 0.1–100 µg/ml ACN in rat hepatocytes (Williams et al. 1985)	UDS responses in cultured rat hepatocytes were negative at doses up to 10 mg/ml ACN or 10 mM or 1.0 mM ACN or CNEO respectively	Probst and Hill 1985; Butterworth et al. 1992; Williams et al. 1985
	0.1 mM CNEO in human mammary cells	UDS was observed in human mammary cells exposed to CNEO but not ACN	Butterworth et al. 1992
DNA repair: UDS autoradiography (in vivo)	60–75 mg/kg ACN by gavage in rats	A single study of UDS in rats receiving ACN 60 or 75 mg/kg by gavage was negative	Butterworth et al. 1992
DNA repair: SCE (in vitro)	0.0000001–0.0001 M ACN in CHO cells (Ved Brat and Williams 1982) 0.0005 M AN in human lymphocytes (Perocco et al. 1982) up to 50 µg/ml ACN in CHO cells (Gulati et al. 1985) 2 mM ACN in CHO cells (Natarajan et al. 1985) 150–300 µg/ml ACN in human bronchial epithelial cells (Chang et al. 1990)	Positive SCE responses were observed in cultured rodent and human cells and human PBLs exposed to ACN at doses in the 10-3–10-4 range	Ved Brat and Williams 1982; Perocco et al,. 1982; Gulati et al. 1985; Natarajan et al. 1985; Chang et al. 1990
	1.25–10 µg/ml ACN in rat liver cells (Priston and Dean 1985) 1–10 µg/ml ACN in human PBL (Obe et al. 1985)	SCE responses were not observed in rat liver cells or human PBLs exposed to somewhat lower doses of CAN	Priston and Dean 1985; Obe et al. 1985
DNA repair: SCE (in vivo)	10–45 mg/kg ACN in mice (Sharief et al. 1986) 7.5–10 mg/kg ACN ip in mice (Fahmy 1999)	SCE responses were induced in mice administered ACN by i.p injection at doses of 10 or 45 mg/kg	Sharief et al. 1986; Fahmy 1999

Table 2. ACN/CNEO associated specific DNA adducts.

Endpoint	Methods	Results, comments	Reference
ACN/CNEO specific adducts, isolated DNA/nucleobases (in vitro)	1 mM ACN, 100 mM CNEO in calf thymus DNA (Guengerich et al. 1981) 1.4 M ACN (Solomon et al. 1984) 50 mM CNEO (Solomon et al. 1993, 1994) 150 mM CNEO (Yates et al. 1993) 100 µM CNEO (Walker, Fennell, et al. 2020)	Adducts formed with all four nucleobases and phosphate backbone exposed to ACN/CNEO, generally under non-physiological conditions	Guengerich et al. 1981; Solomon et al. 1984, 1993, 1994; Yates et al. 1993; Hogy and Guengerich 1986; Walker, Fennell, et al. 2020
	0.0001–0.001 M ACN	ACN failed to form a stable guanosine adduct under physiological conditions but did at higher pH values	Manso et al. 2011
ACN/CNEO specific adducts, human TK6 cells (in vitro)	100 µM CNEO	N7OEG adduct not formed in humanTK6 cells exposed for two hours to 100 mM CNEO	Walker, Fennell, et al. 2020
ACN/CNEO specific adducts, rodent (in vivo)	50 mg/kg ACN or 0.6 mg/kg CNEO ip in rats (Hogy and Guengerich 1986)	N7OEG adduct (radiometric) formed in rats after ACN/CNEO 50 or 0.6mg.kg ip. In single study	Hogy and Guengerich 1986
	1.2 ml of 0.2 mM ACN ip in rats (Peter, Appel, et al. 1983) 50 or 100 mg/kg ACN sc in rats (Prokopczyk et al. 1988) 100 ppm ACN in dw in rats (Williams et al. 2017) 3–300 ppm ACN in dw in rats (Walker, Fennell, et al. 2020)	No specific adducts formed (radiometric or HPLC) after s.c. or oral ACN up to 100 mg/kg in rats in four studies.	Peter, Appel, et al. 1983; Prokopczyk et al. 1988; Williams et al. 2017; Walker, Fennell, et al. 2020
ROS induced adducts, direct chemical determination (in vitro)	0.01–1 mM ACN in rat astrocytes (Kamendulis, Jiang, Xu, et al. 1999) 25–75 µg/ml ACN in CHO (Zhang et al. 2002) 200–400 µM can in NHAs (Jacob and Ahmed 2003) 0.1–1 canACN in rat astrocytes (Klaunig and Forney 2010)	8xoG signature oxidative DNA adduct formcanin ACN treated rat or human astrocytes or Syrian hamster embryo cells at 75–1000 µM range	Kamendulis, Jiang, Xu, et al. 1999; Jacob and Ahmed 2003; Zhang et al. 2000, 2002; Klaunig and Forney 2010
ROS induced adducts, direct chemical determination rodents (in vivo)	30–300 ppm ACN in dw in rats (Whysner et al. 1998) 0–200 ppm CAN in dw in rats (Jiang et al. 1998; Pu et al. 2009, 2015)	8oxoG adducts formed in dose dependent manner in several cells of rats treated orally with ACN at doses from ∼30 to 100 mg/kg.(2)	Whysner et al. 1998; Jiang et al. 1998; Pu et al. 2009; Pu et al. 2015
	2.5–20 mg/kg ACN in mice	No 8oxoG adducts induced in mice.	Kamendulis et al. 2001
ROS induced adducts, modified comet assay (in vitro)	1 mm ACN in rat or human astrocytes (Pu et al. 2006, 2009) 0.1–1 mM ACN in rat astrocytes (Klaunig and Forney 2010)	Positive FPG-G comets were induced in rat astrocytes in vitro by ACN at ∼1.0 mM	Pu et al. 2006, 2009; Klaunig and Forney, 2010
	1 mm ACN in rat astrocytes (Pu et al. 2009)	ACN failed to induce FPG-G comets in human astrocytes	Pu et al. 2009
ROS induced adducts, modified comet assay (in vivo)	3–200 ppm ACN in dw in rats (Pu et al. 2009, 2015) 100 ppm ACN in dw in rats (Williams et al. 2017)	Positive FPG-G comets, various cells, various doses, in rats.(2)	Pu et al. 2009, 2015; Williams et al. 2017
	100 ppm ACN in dw in rats (Williams et al. 2017)	hOGG1 comets negative in tissues of ACN treated rats	Williams et al. 2017

Table 3. Summary of ACN induced somatic level mutations in multiple systems.

Organism and endpoint	Results, comments	Supplemental table
E. coli; trpE reversion, AT base pair	- Most studies positive with external metabolic activation	Table S1
Salmonella reversion (Ames)	- Studies with hisG46 allele (reversion by any one of five base substitutions in CCC) almost universally positive. - Mixed results but mostly negative with other tester strains; usually require external metabolic activation.	Table S1
Fungi, multiple strains	- Mixed results but mostly negative for gene mutations (several target genes); - mostly positive for chromosome level events; most studies of aneuploidy were negative; - usually require external metabolic activation; DEL assay (for detection of oxidative damage) was positive.	Table S2
Mammalian Cells in vitro	- L5178Y mouse lymphoma assay mostly positive for Tk forward mutations with external metabolic activation; - negative for ouabain mutation; - human TK6 cells positive for HPRT forward mutations with external metabolic activation or use of CNEO; - positive results for gene mutations in other cell lines, usually with external metabolic activation; mostly positive results for chromosome level events in cell lines with external metabolic activation but negative in human PBLs (without EMA); - all in vitro studies for aneuploidy were negative.	Table S3
In vivo mutations in mammals	- Hprt mutations increased in lymphocytes in both rats and mice after ACN administered by gavage; - lacZ mutations not increased in any of several tissues of transgenic mice receiving ACN in drinking water; - 5 in vivo studies of chromosome level events were negative in either mice or rats, 2 studies showed weakly positive results, and 4 showed positive results.	Table S4
Human studies	- Increased frequencies of HPRT T-cell mutations in industrial workers detected by autoradiography; - complex exposures with illness in worker populations; - mixed results of induced chromosome level events with positives and negative studes; - reports of ACN-associated mitochondrial changes.	Table S5

Table 4. ACN genotoxicity/mutagenicity in germinal tissues.

Study	Endpoint	Methods	Report	Comment	Reference
Isolated rat and human testicular cells (in vitro)	DNA ss breaks	30–1000 µM ACN; Alkaline elution	Negative	Relevance?	Bjorge et al. 1996
Rats (in vivo)	DNA (protein?) binding	46.5 mg/kg ACN by gavage; Radioactivity	Positive	Not in germ cells; protein contamination	Ahmed, Abdel-Rahman, et al. 1992
	UDS	46.5 mg/kg ACN by gavage; scintillation spectroscopy	Positive	Not in germ cells; possible SDS	Ahmed, Abdel-Rahman, et al. 1992
	UDS	60–75 mg/kg ACN by gavage in rats; autoradiography in spermatocytes	Negative	True negative in germ cells	Butterworth et al. 1992
Mice, wild type (in vivo)	DNA ss breaks in ovarian tissue	10–60 mg/kg ACN gavage in mice; Alkaline comet	Negative	Not in germ cells	Walker, Walker, et al. 2020
Mice/transgenic (in vivo)	DNA ss breaks in ovarian tissue	10–60 mg/kg ACN gavage in mice; Alkaline comet	Positive	Not in germ cells, artifact?	Walker, Walker, et al. 2020
Drosophila (in vivo)	Sex chromosome aneuploidy in offspring	2.7 ppm ACN in air	Positive	Positive for loss only	Osgood et al. 1991
	Sex linked recessive lethal mutations	0.1% ACN solution injected	Negative	Negative	Benesh and Shram 1969
	Sex linked recessive lethal mutations	420 ACN ppm feed; 3500 ppm ACN injected	Negative	Negative	Foureman et al. 1994
Mice (in vivo)	LacZ mutations in testicular tissue cells	10–60 mg/kg ACN gavage in mice	Negative	Not in germ cells, insensitive	Walker, Walker, et al. 2020
	Chromosome aberrations/ spermatogonia	20 or 100 mg/m3 ACN in air	Negative	Negative	Zhurkov et al. 1983
	Chromosome aberrations/ spermatocytes	7.75–31 mg/kg ACN ip in mice	Positive	Unknown significance of univalent	Fahmy 1999
	Dominant lethal test	30 mg/kg ACN ip in mice	Negative	Negative	Leonard et al. 1981
	Dominant lethal test	20 or 100 mg/m3 ACN in air	Negative	Negative	Zhurkov et al. 1983
	Dominant lethal test	60 mg/kg/day ACN oral for 5 days	Negative	Negative	Working et al. 1987
Human (in vivo)	DNA ss breaks in sperm	ACN production workers (predominantly via inhalation); Alkaline comet	Positive	Multiple concerns (see text)	Xu et al. 2003
	Sex chromosome aneuploidy	ACN production workers (predominantly via inhalation)	Positive	Multiple concerns (see text)	Xu et al. 2003

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