Table 1.

Acrylamide (AA) exposure-induced toxicity.

Animal/cell lines	Route of administration/dosage	Biological samples/techniques/parameters assessed	Findings	Inference	Reference
Male wild-type (CYP2E1+/+) vs. CYP2E1-null mice (C57DL/6N2 × 129Sv), 3–4 months old	AA (0–50 mg/5 mL saline/kg), intraperitoneal	Plasma AA and GA levels; red blood cells—hemoglobin adducts; livers, lungs, testes—DNA adducts (LC–ES/MS/MS analysis)	AA produced significant increase in N7-GA-Gua and N3-GA-Ade adducts in all tissues of WT-mice. Significantly less adducts were detected in liver, lungs, and testes of mutant mice. Significant increase in AAVal-PTH and GAVal-PTH adducts in WT-mice. Ratio of GAVal-PTH: AAVal-PTH in AA-treated WT-mice was 1.7, the ratio was 0.02 in null mice	CYP2E1 is the main enzyme involved in epoxidation of AA to GA, which is responsible for formation of GA-DNA and hemoglobin adducts	(38, 39)
Human ovarian cancer cell lines PA-1, SK-OV-3, EF-27; human endometrial cancer cell lines MFE-319. FE-194; human primary hepatocytes	Incubation of cell lines with 0.02 and 1 mM AA and GA for 72 h	Microarray analysis, real-time PCR, Western blot analysis (genes or proteins involved in cellular stress, oncogenesis, xenobiotic metabolism)	Upregulation of the genes involved in angiogenesis such as TF3 (ovarian and endometrial cell lines), dual specific phosphatases DUSP1, DUSP4, and DUSP5 (ovarian cell lines), zinc finger protein gene ZNF746, cYMC and Bystin-like protein gene (BYSL) (primary human hepatocytes)	High dose of AA can induce genes with growth promoting potential like oncogene cMYC and genes involved in MAPK pathway	(41)
Male F344 rats	AA was administered in the drinking water at concentrations of 2.5, 10, and 50 mg/kg bw/day for 14 days	Motor activities; serum levels of thyroid-releasing hormone, thyroid-stimulating hormone (TSH), thyroid hormones (T3, T4); target tissue expression of genes involved in hormone synthesis, release, and receptors; dopamine and serotonin levels in hypothalamus and pituitary gland; and histopathological evaluation of the pituitary glands	50 mg/kg/day AA caused lethargy and hind-limb paralysis; 50 mg/kg/day AA significantly decreased serum T4 levels. No significant effects on other parameters assessed	Sub-chronic treatment of F344 male rats with AA provided no evidence for anti-thyroid effects manifested by compensatory increases in cell proliferation through dysregulation in the hypothalamus or pituitary	(49)
Male F344/DuCrl rats	0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw/day was given in drinking water for 5, 15, or 31 days	Expression profiling by next-generation sequencing of RNA, bioinformatics to identify differentially expressed genes and gene ontology pathway analysis, and qPCR was performed on testes samples	The largest number of differentially expressed genes (DEG) (65 transcripts) was observed at the highest AA exposure level (12.0 mg/kg/day) on day 31. 6.0 and 12.0 mg/kg of AA significantly increased Cyp2a1 rat testosterone 7 a-hydroxylase; QPCR showed significant increase in Arsi at 0.5, 1.5, 6.0, and 12.0 mg/kg, Cacnb1at 1.5 and 6.0 mg/kg, Cacng7 at 0.5 and 12.0 mg/kg, S100b at 0.5, 1.5, 3.0, 6.0, and 12.0 mg/kg, Tnnt2 at 12.0 mg/kg, Trpc1 at 1.5 and 3.0 mg/kg (p ≤ 0.05)	AA via calcium signaling and cytoskeletal actin filaments can cause rat dominant lethal mutations that could lead to impaired chromosome segregation during cell division	(53)
Bovine eyes (3–4 years old)	Incubation of cell lines with staurosporine with/without 5 mM AA for 8 or 24 h; cells were preincubated with AA 5 mM in serum-containing medium; cells were incubated with AA 5 mM alone	Indirect immunofluorescence on antibodies against vimentin and human α-tubulin; percentage of apoptosis was calculated upon exposure to AA	AA exposure caused collapse of vimentin and microtubules paralleling cytoskeletal disruption; Cell adopted nearly a rounded morphology; thick f-actin bundles remaining in the cell periphery; AA slightly increased apoptosis compared to controls. Simultaneous exposure to AA and staurosporine for 8 h produced significantly less apoptosis and preincubation with AA followed by staurosporine reduced apoptosis at 8 and 24 h of treatment	AA exerts significant effects on the cytoskeleton of bovine eyes cells and AA can significantly attenuate the apoptotic effect of staurosporine	(56)
Male F344/DuCrl rats (8 weeks old)	Rats were given 0.0, 0.5, 1.5, 3.0, 6.0, or 12.0 mg AA/kg bw day in drinking water for 5, 15, or 31 days	Serum analysis of hormones [triidothyronine (T3), thyroxine (T4), reverse T3, TSH, lutenizing hormone, prolactin, and testosterone]; Ion Proton™ sequencing (RNA-seq) was done to calculate DEG and qPCR to quantitate gene expressions in liver and thyroid samples	6.0 mg/kg bw day AA for 31 days significantly increased the absolute and relative thyroid weights; five 5 days of exposure to 1.5 mg/kg bw day AA significantly decreased serum T3 levels. T4 levels were significantly increased on day 5 at the highest dose, and on day 31; gene expression in liver: high dose of AA affected gene expression of Brca1, Top2a, Cenpf, Nr1d1; gene expression in thyroid: Nqo1 and Hmox1 was differentially expressed at the highest dose on day 31, increased expression of Cyp2e1 on day 31 following exposure to 0.5 and 12 mg/kg bw day AA, genes involved in cytoskeletal pathways were differentially expressed (Acta1, Atp2a1, Myl1, Myh1, and Pvalb)	Mechanism of action for AA-induced thyroid carcinogenicity in male rats may due to perturbation of calcium signaling	(55)
Male Sprague-Dawley rats, 8 weeks old	Experiment 1: animals were gavaged (0, 30, 45, and 60 mg/kg/bw of AA) one a day for five consecutive days; Experiment 2: animals were gavaged single dose of 125, 150, and 175 mg/kg/bw of AA	Histochemical demonstration of alpha-naphthyl acetate esterase (ANAE) was done on blood and lymphoid tissue samples from the ileal Peyer’s patches (IPP)	Histopathology: 125, 150, and 175 mg/kg/bw of AA significantly reduced IPP size, depleted lymphoid cells in follicles of IPP, regression and reduction of germical left’s size; ANAE histochemistry: ANAE-positive lymphocyte levels decreased significantly (p < 0.05) in all AA received animals in a dose-dependent manner—highest decrease seen in 45 and 60 mg/kg/bw (Experiment 1), highest decrease seen in 150 and 175 mg/kg/bw (Experiment 2)	Exposure to AA is detrimental to peripheral blood lymphocytes and gut-associated lymphoid tissues in rats	(91)
Female BALB/c Mice (6–8 weeks old) of specific pathogen-free grade	4, 12, 36 mg/kg/bw of AA dissolved in distilled water given for 30 days	Assessment of body weight, spleen and thymus weight, hematological parameters, phenotyping of peripheral blood samples, quantification of serum cytokines and immunoglobulin, hemolysis test; plaque-forming cell assay and mitogen-induced splenocyte proliferation and splenic natural killer (NK) activity was assessed using mice splenic suspension; histopathology of various organs was studied	AA exposure significantly reduced body weight, spleen weight and thymus weight of mice; AA significantly increased percentage of T lymphocytes (CD3+, CD19−), CD4+ T lymphocytes (CD3+, CD4+, Th cells); AA significantly decreased percentage of NK cells (CD3−, CD49+); AA significantly reduced serum IL-6 level; AA significantly reduced the HC50 and the ConA-induced splenocyte proliferation; histopathology: mild atrophy of thymus, decrease in the number of bone marrow (hematopoietic) stem cells, shrinking of lymphoid nodules in germinal left in spleen, and white pulp atrophy of spleen, lymphopenia, and proliferation of fibrous tissue in lymph glands, unclearness or disappearance of follicle structure, and atrophy of lymph glands	AA inhibited cellular and humoral immunity of mice following 30 days of exposure	(92)
C57BL/6 male mice (young 7 weeks; aged 18−20 months)	RAW 264.7 cells and peritoneal macrophages were exposed to AA (<1 mM)	Macrophages tested in senescence associated-β-galactosidase (SA-β-gal) assay, telomerase repeat amplification protocol (TRAP) assay, cell cycle analysis, ROS production assay, ATF3 protein, and gene expression studies	AA (<1 mM) caused senescence-like growth arrest, significantly increased protein and gene expression of ATF3; intracellular ROS levels significantly increased in a time-dependent manner at 0.25 and 0.5 mM ACR; AA-induced macrophage senescence via the p38 and JNK pathways mediated by ATF3	ACR-induced senescence aided by ATF3 by enhancing ROS production, activating p38 and JNK kinases, and upregulating the expression of p53, collectively leading to cellular senescence in macrophages	(93, 94)
Mouse Leydig (TM3) and Sertoli (TM4) cell lines derived from the testis of immature BALB/c mice (11–13 days old)	Cell lines exposed to AA (10 and 1 mM) and GA (1 and 0.5 mM) for 24 h	Leydig and Sertoli cells were evaluated for cell viability, lactate dehydrogenase activities, lipid peroxidation, H2O2, apoptosis/necrosis rate, and mRNA levels of apoptotic genes	High AA and GA doses significantly decreased viability of Leydig cell Sertoli cells (p < 0.01); dose-dependent decrease in LDH activities of Leydig and Sertoli cells following AA and Ga exposure (p < 0.01); Leydig cells: significant increase in MDA levels in the all AA- and GA-treated groups. Sertoli cells: increase in MDA levels only by high AA and GA doses (p < 0.001); significant increases in H2O2 levels following AA and GA exposure in Leydig and Sertoli cells; 10 mM AA exposure of TM3 cell line significantly increased the expression of caspase3 (2.6-fold), Bax (1.3-fold), and p53 (1.1-fold) and decreased Bcl-2 (0.5-fold); 1 mM of AA increased the expression of caspase3 (2.5-fold), Bax (2.3-fold), and p53 (2.5-fold) and decreased Bcl-2 (0.3-fold); exposure of 1 mM GA to TM3 cell line, significantly increased caspase3 (2.1-fold), Bax (2.2-fold), and p53 (1.4-fold) and decreased the expression of Bcl-2 (0.5-fold) gene expression; GA (0.5 mM) increased caspase3 (3.6-fold), Bax (4.6-fold), and p53 (4.9-fold) and decreased Bcl-2 (0.2-fold)	Oxidative stress could play central role in AA and GA-induced apoptosis of Leydig and Sertoli cells	(84)
Adult male Sprague-Dawley rats	exposed to ACR at daily dose-rates of either 50 mg/kg/day (ip) for 5 days or 21 mg/kg/day (po) for 21 days	Body weight and gait score was determined; striatal synaptic vesicles from the rats were used to test for vesicular 3H-dopamine uptake and spontaneous efflux and density of degeneration was determined using amino-cupric silver staining and light microscopic examination; synaptosomal dopamine uptake and release following AA exposure (in vivo) was assessed; synaptosomal gluthathione content was assessed following AA exposure (in vivo)	50 mg/kg of AA reduced the body weight and increased the gait score; 21 mg/kg AA increased the gait score; exposure to 50 mg/kg/day × 5 days or 21 mg/kg/day × 21 days significantly decreased vesicular DA uptake and also reduced KCl-evoked synaptosomal DA release; AA did not affect neurotransmitter retention	AA impaired neurotransmitter uptake into striatal synaptic vesicles through interaction with sulfhydryl groups on functionally relevant proteins which leads to defective presynaptic release	(78)