Table 3.

Studies on in vitro and in vivo DNA adducts induced by AA and/or GA

Test	Analytical method	Experimental design and doses	Results	Comments	Reference
In vitro
DNA adducts (N7‐GA‐Gua and N3‐GA‐Ade) in calf thymus DNA	LC–MS	GA: 10,000, 25,000, 50,000 µM	Positive GA At neutral conditions DNA adducts are released from DNA (spontaneous depurination)	GA‐induced lesions are predominantly N7‐GA‐dG adducts slowly undergoing imidazole ring opening at pH 10; such structures are incised by Fpg leading to DNA strand breaks. See Table 4 for further information.	Hansen et al. (2018)
DNA adducts (N7‐GA‐Gua and N3‐GA‐Ade) in (Hupki) mouse embryo fibroblasts	LC–MS/MS LOD: 5.5 adducts per 108 nucleotides	AA: 5,000 µM + 2% human S9 AA: 10,000 µM −S9 GA: 3,000 µM Treatment time: 24 h	AA −S9: no N7‐GA‐Gua adducts AA +S9: 11 adducts per 108 nucleotides GA: 49,000 and 350 adducts per 108 nucleotides for N7‐GA‐Gua and N3‐GA‐Ade, respectively. Control: 5.5 adducts per 108 nucleotides	Mutations by whole exome sequencing in (Hupki) mouse embryo fibroblasts (Negative AA; Positive GA).	Zhivagui et al. (2019)
DNA adducts (N7‐GA‐Gua) in (Hupki) mouse embryo fibroblasts	UPLC–ESI‐MS/MS LOQ: 1.7 adducts per 108 nucleosides	AA: 0, 1,000, 1,500, 3,000 µM (48 h) GA: 0, 750, 1,100, 1,500 µM (24 h)	Negative AA: < LOQ Positive GA: 20 (at 750 and 1,100 µM) and 30 (at 1500 µM) N7‐GA‐Gua per 108 nucleosides; below LOQ at 48 h; no adducts in control.	Mutations by whole genome sequencing in (Hupki) mouse embryo fibroblasts (Negative AA; Positive GA). See Table 4 for further information.	Hölzl‐Armstrong et al. (2020a)
DNA adducts (N7‐GA‐Gua) in rat hepatocytes		AA: 2, 20, 200, 500, 1,000, 2,000 μM Treatment time: 16 and 24 h	Positive AA: non‐linear concentration response; increases in N7‐GA‐Gua at 1,000 and 2,000 μM (20–30 adducts per 108 nucleotides); background levels: 5–10 adducts per 108 nucleotides		Hemgesberg et al. (2021a)
In vivo
DNA adducts (N7‐GA‐Gua and N3‐GA‐Ade) in lung and liver of B6C3F1 mice (F) (5‐6 weeks of age)	Analysis by HPLC–ESI‐MS/MS LOD: 0.5 adducts per 108 nucleotides for both N7‐GA‐Gua and N3‐GA‐Ade LOQ: 1 and 1.5 adducts per 108 nucleotides for N7‐GA‐Gua and N3‐GA‐Ade, respectively	Oral (drinking water) AA: 0, 87.5, 175, 350, 700 µM (2‐year cancer bioassay concentrations) (equivalent to 1.04, 2.20, 4.11 and 8.93 mg/kg bw per day for M, and to 1.10, 2.23, 4.65 and 9.96 mg/kg bw per day for F) Treatment time: 28 days	Positive AA: dose‐dependent increase in both DNA adducts in lung and liver. Similar levels of adduction in the two organs. N7‐GA‐Gua: range 100–1,000 N3‐GA‐Ade: range 0.5–4 per 108 nucleotides	Important role of epigenetic alterations in determining the target organ for AA tumorigenesis. See Table 4 for further information.	de Conti et al. (2019)
DNA adducts (N7‐GA‐Gua) and GAMA3 in urine and tissues (liver, kidney and lung) of Sprague‐Dawley rats (F) (7‐8 weeks of age, 12 animals/dose group)	UPLC–MS/MS LOD and LOQ for N7‐GA‐Gua: 1 and 3 adducts per 108 nucleotides (0.02–0.06 pmol, respectively).	Gavage AA: 35 mg/kg bw per day Treatment time: 7 and 14 days Urine collection: 0 (0.5 h), 0.5 (1 h), 1 (2 h), 2 (4 h), 4 (8 h) following 1st AA gavage, sacrifice at 14th days.	Positive N7‐GA‐Gua adducts per 108 nucleotides at 7 and 14 days, respectively: – Liver: approx. 900 and 1,300 – Kidney: approx. 1,300 and 2,100 – Lung: approx. 1,000 and 1,900 Pre‐treatment with blueberry anthocyanin extract: significant block of AA epoxidation to GA.		Wang et al. (2019)
DNA adducts (DNA N7‐GA‐Gua) and AAMA in urine of smokers (n = 30) and non‐smokers (n = 33)	Isotope dilution SPE LC–MS/MS LOD for N7‐GA‐Gua: 0.25 ng/mL in urine	–	Positive: median urinary N7‐GA‐Gua level: 0.93 and 1.41 μg/g creatinine (1.50 and 2.01 ng/mL urine) in non‐smokers and smokers, respectively (this difference is not statistically significant). Urinary N7‐GA‐Gua levels significantly associated with AAMA levels.	The authors conclude that urinary N7‐GA‐Gua of non‐smokers and smokers is significantly associated with a very low level of dietary AA intake.	Huang et al. (2015)
DNA Adducts (DNA N7‐GA‐Gua) and mercapturic acid derivatives AAMA and GAMA in the urine of 8 male AA‐exposed workers and 36 controls (administrative workers) from the same AA production plants (four plants). Collection of urines pre‐ and post‐shifts at work	Analysis of DNA N7‐GA‐Gua by isotope‐dilution two‐step SPE LC–MS/MS Analysis of AAMA and GAMA: LC–ESI‐MS/MS LOD of N7‐GA‐Gua: 0.25 ng/mL in urine (50 fmoles)	‐	Positive: Significantly higher levels in AA‐exposed workers than in controls at both time points; Median urinary N7‐GA‐Gua level in exposed workers: 1.85 ng/mL and 2.26 ng/mL (at pre‐ and post‐shift, respectively). Median urinary N7‐GA‐Gua level the control group: 0.33 ng/mL and 0.29 ng/mL (at pre‐ and post‐shift, respectively). The elimination half‐life of urinary N7‐GA‐Gua was 346.5 h (140.3–737.2 h). N7‐GA‐Gua level correlated positively with AAMA and GAMA levels.	The two groups had similar BMIs, periods of employment, smoking status and preferences for fried food.	Huang et al. (2018)
DNA adducts (N7‐GA‐Gua) in PBMC from 56 healthy volunteers (age 18–65 years)	UHPLC–ESI+‐MS/MS LOD: 0.1–0.2 adducts per 108 nucleosides LOQ: 0.3–0.5 per 108 nucleosides	Measurements of biometric, dietary and biochemical parameters: (age, gender, body weight, height, smoking, vegetarian, vegan, frequent consumption of coffee, French fries, cookies)	N7‐GA‐Gua DNA adducts: mean: 1.1 per 108 nucleosides; range: 0.2–26.6 per 108 nucleosides Correlation with BMI. No correlation with dietary habits, blood glucose levels and haemoglobin.		Hemgesberg et al. (2021b)
DNA adducts (N7‐GA‐Gua) in blood samples of 17 healthy volunteers	LC–ESI‐MS/MS LOD: 25 fmoles LOQ: 0.50 fmoles		Positive: DNA adducts quantified in 13 out of 17 samples N7‐GA‐Gua range: 0.3–6.3 adducts per 108 nucleotides N7‐GA‐Gua mean: 1.9 adduct per 108 nucleotides	Range of AA intake from the food frequency questionnaires: 20.0–78.6 μg per day (corresponding to 0.29–1.14 µg/kg bw per day, using the mean body weight of 69 kg provided by the authors)	Jones et al. (2021)

AA: Acrylamide; AAMA: N‐acetyl‐S‐(2‐ carbamoylethyl)‐L‐cysteine; BMI: body mass index; F: females; Fpg: DNA‐formamidopyrimidine glycosylase; GA: glycidamide; GAMA3: N‐acetyl‐S‐(3‐amino‐2‐hydroxy‐3‐oxopropyl)‐cysteine; GAMA: N‐(R,S)‐acetyl‐S‐(1‐carbamoyl‐2‐hydroxyethyl)‐l‐cysteine; 8‐OHdG: 8‐hydroxy‐2'‐deoxyguanosine; LOD: limit of detection; LOQ: limit of quantification; M: male; N3‐GA‐Ade: N3‐(2‐carbamoyl‐2‐hydroxyethyl)adenine; N7‐GA‐Gua: N7‐(2‐carbamoyl‐2‐hydroxyethyl) guanine; LC–MS/MS: liquid chromatography tandem mass spectrometry; PBMC: peripheral blood mononuclear cell; SPE: solid‐phase extraction; UHPLC–ESI‐MS/MS: ultra‐high‐performance liquid chromatography‐electron spray ionisation‐tandem mass spectrometry.