Neurobehavioral alterations and histopathological changes in brain and spinal cord of rats intoxicated with acrylamide

Abstract

The aim of this project was to study the clinical manifestations, neurobehavioral, hematobiochemical, oxidative stress, genotoxicity, and histopathological changes during acrylamide toxicity in rats. A total of 30 adult male Wistar rats were divided in 5 equal groups and received 0, 10, 15, and 20 mg/kg body weight acrylamide as oral gavage, while group 5 was micronucleus (MN) control. Functional observational battery (FOB) parameters were studied at the 28th day of post treatment. Toxicological manifestations were evident in acrylamide-treated rats from 14th day onward. FOB revealed a significant change in central nervous system, neuromuscular, and autonomic domains. The hematological changes include significant decrease in concentration of hemoglobin, total erythrocyte count, packed cell volume, and mean corpuscular volume. The biochemical parameters aspartate aminotransferases, alkaline phosphatase, and albumin showed significant increase, while the levels of serum globulin and glucose were found to decrease significantly. The MN assay revealed the significant increase in frequencies of micronuclei and number of polychromatic erythrocytes. The oxidative stress parameters revealed no significant difference as compared to control rats. Histopathological changes observed in brain include neuronal degeneration, edema, and congestion, while spinal cord revealed demyelination in low-dose group and bilateral necrosis with malacia, liquefaction of white matter, and loss of myelin from gray matter in high-dose groups. The result indicates pathological alterations in brain and spinal cord and is responsible for neurobehavioral changes in rats. The FOB changes and histopathological alterations in spinal cord are in dose dependent to acrylamide intoxication. Various toxicological effects observed in experiment direct us to focus on a deep study and evaluate the possible causes pertaining to toxicity of this chemical. It would furnish the scientists with better options that would help them to search for a median path regarding the use of this chemical and take preventive measures to save the living beings from the hidden disasters of this chemical.

Keywords

Acrylamide toxicity genotoxicity neurotoxicity oxidative stress histopathology

Introduction

Acrylamide is found in carbohydrate-rich food prepared at high temperatures such as French fries and potato chips consumed by humans. Consumption of these foods may result in significant human exposure to acrylamide (Tareke et al., 2002). Subsequent biochemical analysis identified the origin of the dietary acrylamide by demonstration that the heating of free amino acids, in particular asparagines and sugars during food processing (120–180°C) results in the formation of acrylamide (Mottram, 2002). The two major forms of acrylamide are monomer and polymer. Monomer form is highly toxic, while the polymer form is reported to have very low toxicity or no toxicity (Tyl and Crump, 2003; Vattem and Shetty, 2003). The largest use of polyacrylamide is in treating municipal drinking water and industrial waste water to remove suspended solids before discharge, reuse, or disposal (Croll et al., 1974). Polyacrylamide polymers and copolymers are used as binders in the article and textile industries, as soil conditioners, in ore processing, and in cosmetics (Friedman, 2003). These also play various roles in photography, dyeing, electrophoresis, animal feed, pharmaceuticals, adhesives, tapes, and gels (Giese, 2002).

Early human epidemiological and laboratory animal studies indicated that acrylamide exposure produces skeletal muscle weakness and ataxia (LoPachin and Lehning, 1994), reduced light sensitivity, and reduced color discrimination (Goffeng et al., 2008). Continued research suggested that behavioral neurotoxicity is a product of nerve damage classified as central peripheral distal axonopathy (Spencer and Schaumburg, 1977). Acrylamide is now recognized as the prototype chemical for a class of compounds that produce distal axonal degeneration. A study indicated that acrylamide administration could produce classical behavioral neurotoxicity without any indication of altered axon morphology in peripheral nerves (Lehning et al., 1998). Acrylamide exposures also produce measurable decrement in food-motivated behavior (Garey and Paule, 2007). Acrylamide can undergo oxidative biotransformation by cytochrome P450 (CYP) 2E1 (Sumner et al., 1999). The resulting metabolite is an epoxide derivative, that is, glycidamide, which is more reactive toward DNA and proteins than the parent compound, acrylamide (Dearfield et al., 1995). The biological consequences of acrylamide exposure have chiefly centered on neurotoxicity ever since this effect was observed in humans occupationally exposed to this compound (McCollister et al., 1964). Subsequently, experimental exposure of rodents to acrylamide has also revealed a carcinogenic mode of action for this chemical (Friedman, 2003). The fate of acrylamide seems to be qualitatively similar in mammalian species examined more extensively to date, including mice, rats, and humans.

The present study outlines a validation strategy emphasizing the characterization of a behavioral profile of acrylamide toxicity and its relation with damage in spinal cord and brain. Also, present experiment was conducted to investigate the dose-dependent toxicity of acrylamide on hematobiochemical, lipid peroxidation (LPO), and pathological findings in various organs and genotoxicity in male Wistar rats.

Materials and methods

Animals

The animal experiment was carried out according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the local authorities. All efforts were made to minimize the number of animals used and their suffering. Acrylamide (99.50% purity) was purchased from Hi Media Laboratories Pvt. Limited (Mumbai, Maharashtra, India), and all other required chemicals were used of extra pure grade. A total of 30 male Wistar rats were obtained from the Shree Farms (Bhandara, Maharashtra, India). The rats weighted 150 ± 10 g and were housed under conventional conditions at room temperature of 21–24°C with a 12-h light: 12-h dark cycle and humidity of 50–60%. Experimental rats were divided randomly into five treatment groups (A, B, C, D, and E) with six rats per exposure group: groups A, B, C, and D were administered via oral gavages for 28 days with acrylamide at 0, 10, 15, and 20 mg/kg body weight (b.w.), respectively, as single dose daily in distilled water. Animals from group A served as control. Rats in group E were administered with cyclophosphamide at 20 mg/kg body weight intraperitoneally for 24 h prior to killing and served as positive control for genotoxicity study. All animals were observed daily for any abnormal physical or behavioral changes. The time of onset, intensity, and duration of such toxic manifestations, if any, was recorded till 28 days.

Neurobehavioral FOB

During the course of study, the neurobehavioral functional observational battery (FOB) was developed as a simple and objective way to screen for preliminary disturbances in the neurobehavioral make up of animals. The FOB parameters were studied as per protocols laid down (Youssef and Santi, 1997). The FOB consisted of side cage observation (30 s); open field observation (2 min); and evaluation of handling, reflexes, gait scoring, and grip strength of the forelimbs and hind limbs. Temperature and b.w. were measured. Gait analysis was based on the measurements of the distances and angles between the sequential foot strides after walking in a corridor. Landing foot splay was the distance between the inner surfaces of the fourth digit of each foot after the animals were dropped from a 30-cm height.

Body weight

The b.w. of each rat was recorded one day before initiation of treatment (day 0) and thereafter at weekly intervals throughout the period of study. The last b.w. was recorded on the day of euthanasia prior to blood collection.

Hematology

Blood samples were collected on the day of euthanasia (day 28) in two vials, one coated with ethylenediaminetetraacetic acid as anticoagulant from retro orbital plexus with the help of capillary tube and in second vial without anticoagulant and was used for serum separation. Hematology parameters like hemoglobin (Hb), packed cell volume (PCV), total erythrocyte count (TEC), total leukocyte count (TLC), differential leukocyte count (DLC), and hematocrit values were studied by standard routine procedures laid down (Benjamin, 2001).

Measurement of serum enzyme levels

Blood samples were collected in vials without anticoagulant and were kept undisturbed. The serum was separated and stored at −20°C for subsequent analysis. Serum amino aspartate transferases (AST), alanine aminotransferases (ALT), alkaline phosphatase (ALP), glucose, total protein, albumin, globulin, creatinine, cholesterol, and triglyceride estimations were carried out by analytical kits supplied by Ranbaxy Fine Chemical Limited (Haridwar, Uttarakhand, India), on clinical chemistry semi autoanalyzer.

Postmortem examination

For postmortem examination and collection of tissue samples, animals were killed by cervical dislocation at the end of experiment. The detailed postmortem examination was carried out. Detailed postmortem lesions from all the animals were recorded. For gross (macroscopic) lesions, liver, kidney, lung, heart, and spleen were collected and examined. The lungs, liver, kidneys, heart, spleen, and brain were collected for measurement of weight. After collection, organs were cleaned using filter paper and then weighed using analytical balance (Sartorius, Bohemia, New York, USA). The weights of all the organs were recorded as absolute weights.

Histopathology

The representative tissue samples of liver, kidney, heart, spleen, brain, and spinal cord were fixed in 10% buffered formalin immediately on removal. They were subjected for histopathological examination following standard procedure.

MN assay

Bone marrow cells from both femurs were collected for evaluation of genotoxicity using micronucleus (MN) assay as per protocol laid down (Chauhan et al., 2000). For MN assay 2000 polychromatic erythrocytes (PCEs) per animal were scored to determine MN frequencies and 200 erythrocytes were examined to calculate the ratio of PCEs to normochromic erythrocytes (NCEs).

Oxidative stress

To study oxidative stress, related tissues, such as liver and kidney, were collected in ice-cold containers for various oxidative stress and lipid peroxidation (LPO) parameters estimation, namely, LPO by Rehman (1984), reduced glutathione (GSH) by Sedlak and Lindsay (1968), and superoxide dismutase (SOD) by Marklund and Marklund (1984).

Statistical analysis

The data generated were analyzed with one-way analysis of variance by Snecdecor and Cochron (1994). The level of significance was set at p ≤ 0.05.

Results

Toxic manifestations

Toxic manifestations were recorded daily morning and evening from the starting day of experiment till the final day. On day 15, two animals from group D started showing splaying of hind limbs. It was continued, along with weakness and loss of pain sensation. On day 20, weakness of hind limbs was recorded in two animals from group C. On day 21, splaying of hind limbs along with paralytic signs was noticed in another two animals from group D. In group B, weakness of hind limbs and altered sitting posture in two animals were noticed on day 24 of the experiment. On day 25, dragging of hind limbs was seen in animals from group D, and on the next day, animals from the same group rested on hind limbs and were unable to walk. On day 27, incoordinated movements were seen in the animals from group C. Similar signs were seen on day 28 of the experiment. Animals from control group A did not revealed any toxic manifestations.

Alterations in neurobehavioral FOB

Central nervous system domain in which latency to move recorded in seconds, number of extra lines crossed, freezing time, and numbers of rears from all the treatment groups are presented (Table 1). Neuromuscular domain in which the observations of parameters studied to evaluate neuromuscular domain from all treatment groups is presented (Table 2). Significant (p ≤ 0.05) decrease in angle of stride was observed in group D as compared to group A (Figures 1 and 2). The indicator of foot splay, that is, increase in interheel distance of intoxicated groups did not differ significantly among themselves (Figures 3 and 4). However, statistically significant (p ≤ 0.05) increase was observed in groups B, C, and D as compared to control group A (Figures 3 and 4).

Table 1.

Mean values of CNS domains during FOB in rats exposed to acrylamide for 28 days.^a

Group	Latency to move (s)	Number of squares crossed	Number of extra lines crossed	Freezing time (s)	Number of rears
A	2.83 ± 0.40	4.00 ± 0.00^b	4.00 ± 0.00^b	0.00 ± 0.00^c	9.16 ± 1.32
B	5.83 ± 1.32	4.00 ± 0.00^b	4.00 ± 0.00^b	1.83 ± 0.47^b,c	7.00 ± 0.85
C	16.83 ± 9.04	2.50 ± 0.42^c	4.00 ± 0.00^b	3.00 ± 0.44^b,d	6.33 ± 0.95
D	21.50 ± 5.16	2.33 ± 0.55^c	2.83 ± 0.47^c	4.00 ± 1.12^d	5.00 ± 1.21

CNS: central nervous system; FOB: functional observational battery; ANOVA: analysis of variance.

^aValues are expressed as mean ± SE of six observations; p < 0.05: mean with different alphabets as superscripts differ significantly using ANOVA test. Alphabets are used for vertical comparison.

Table 2.

Mean values of neuromuscular domains in rats exposed to acrylamide for 28 days.^a

Group	Corridor walking			Grip strength (Grading)		Foot splay
Group	Length (cm)	Width (cm)	Angle (degree)	Forelimb	Hind limb	Interheel distance (cm)	Fourth digit distance (cm)
A	12.76 ± 1.20	5.13 ± 0.15^b	99.66 ± 4.91^c	4.00 ± 0.00^d	4.00 ± 0.00^e	8.07 ± 0.44	11.52 ± 0.70^b
B	11.81 ± 1.45	5.43 ± 0.39^b	87.33 ± 7.29^b,c	2.83 ± 0.30^c	2.33 ± 0.33^d	10.45 ± 0.82	14.83 ± 0.94^c
C	13.60 ± 0.92	6.26 ± 0.58^b,c	85.16 ± 9.32^b,c	2.00 ± 0.36^c	1.33 ± 0.42^c	11.16 ± 0.38	15.67 ± 0.38^c
D	10.58 ± 1.04	7.30 ± 0.43^c	67.20 ± 6.87^b	1.16 ± 0.30^b	0.00 ± 0.00^b	13.58 ± 0.41	18.21 ± 0.51^d

^aValues are expressed as mean ± SE of six observations; p < 0.05: mean with different alphabets as superscripts differ significantly using analysis of variance test. Alphabets are used for vertical comparison.

To study autonomic domain, urine pools were recorded (number of times of urination by each animal from each treatment group) and fecal pellets were recorded (number of pellets passed by each animal from each treatment group) during FOB. There was no statistically significant difference in number of urine pools and fecal pellets passed by animals from each treatment group (data not shown). Rectal temperature (°F) was recorded during FOB. The observations of mean ± SE of temperature from all the treatment groups are 96.68 ± 0.38, 97.08 ± 0.84, 97.16 ± 0.48, and 97.40 ± 0.38 (°F), respectively. There was no statistically significant difference in mean rectal temperature of different treatment groups.

Effect of acrylamide on body and organ weights

The observations of mean ± SE b.w. (in grams) of all the treatment groups are presented (Table 3). There was significant reduction in b.w. of high-dose fed group D as compare to the control. The observations of mean ± SE values of organ weight (in grams) of all the treatment groups are presented (Table 4). The observations of mean ± SE values of relative organ weight (in grams) of all the treatment groups are presented (Table 5). When relative weights (per 100 g b.w.) were compared, no statistically significant difference was recorded.

Table 3.

Mean body weights (in grams) of rats exposed to acrylamide for 28 days.^a

Days of experiment	Groups
Days of experiment	A	B	C	D
Day 0	150.83 ± 5.06	150.00 ± 2.58	150.83 ± 2.00	150.00 ± 2.23
Day 7	174.16 ± 6.50	168.33 ± 4.21	167.50 ± 3.81	167.50 ± 5.28
Day 14	193.33 ± 6.79	188.33 ± 6.00	185.00 ± 7.63	183.33 ± 6.81
Day 21	217.50 ± 6.15^b	200.00 ± 5.77^b,c	195.83 ± 9.52^b,c	176.66 ± 14.00^c
Day 28	238.33 ± 7.49^b	203.33 ± 10.54^c	199.16 ± 9.69^c	180.00 ± 13.66^c

Table 4.

Mean organ weights (in grams) of rats exposed to acrylamide for 28 days.^a

Organ	Groups
Organ	A	B	C	D
Lungs	1.388 ± 0.182	1.213 ± 0.079	1.137 ± 0.070	1.319 ± 0.157
Liver	10.327 ± 0.597^b	7.903 ± 0.264^c	7.216 ± 0.466^c	6.856 ± 0.538^c
Kidney	1.605 ± 0.112	1.536 ± 0.089	1.449 ± 0.110	1.382 ± 0.059
Spleen	0.466 ± 0.032^d	0.440 ± 0.023^b,c	0.379 ± 0.010^b,c	0.353 ± 0.030^c
Heart	0.805 ± 0.059	0.714 ± 0.070	0.711 ± 0.044	0.719 ± 0.044
Brain	1.836 ± 0.056^b	1.700 ± 0.064^b	1.642 ± 0.036^b	1.314 ± 0.105^c

Table 5.

Mean values of relative organ weights (per 100 g body weight) of rats exposed to acrylamide for 28 days.^a

Organ	Groups
Organ	A	B	C	D
Lungs	0.584 ± 0.080	0.600 ± 0.038	0.571 ± 0.028	0.775 ± 0.137
Liver	4.351 ± 0.290	3.923 ± 0.178	3.596 ± 0.219	3.860 ± 0.278
Kidney	0.673 ± 0.042	0.755 ± 0.024	0.734 ± 0.062	0.784 ± 0.048
Spleen	0.195 ± 0.013	0.219 ± 0.015	0.192 ± 0.010	0.202 ± 0.024
Heart	0.336 ± 0.018	0.351 ± 0.028	0.360 ± 0.024	0.414 ± 0.050
Brain	0.776 ± 0.044	0.855 ± 0.077	0.837 ± 0.055	0.747 ± 0.076

Hematological alterations

The observations of mean ± SE values of Hb (in gram per deciliter), PCV (%), TEC (×10⁶ cells per microliter), mean corpuscular volume (MCV; fL), mean corpuscular hemoglobin concentration (MCHC; %), and mean corpuscular hemoglobin (MCH; pg) from all the treatment groups are presented (Table 6). When mean observations of values of TLC (×10³ cells per microliter), and DLC (%) from all the treatment groups were compared and no significant difference among the different treatment groups was recorded (data not shown here).

Table 6.

Mean values of hematological parameters in rats exposed to acrylamide for 28 days.^a

Group	Hb (g/dL)	PCV (%)	TEC (×10⁶ cells/µL)	MCV (fL)	MCHC (%)	MCH (pg)
A	13.16 ± 0.86^b	44.5 ± 0.88^c	8.84 ± 0.16^b	50.31 ± 0.64^d	29.58 ± 0.48	14.88 ± 0.33
B	9.26 ± 0.92^d	31.16^c ± 1.27^b	7.34 ± 0.22^d	42.54 ± 1.76^e	29.77 ± 0.68	12.68 ± 0.65
C	8.66 ± 0.91^d,e	28.33 ± 0.91^d	6.62 ± 0.36^d,e	43.32 ± 2.54^e	30.57 ± 0.78	13.33 ± 1.12
D	7.80 ± 0.55^e	25.00 ± 0.85^e	5.78 ± 0.36^e	43.68 ± 1.90^e	31.23 ± 0.32	13.63 ± 0.54

Hb: hemoglobin; PCV: packed cell volume; TEC: total erythrocyte count; MCV: mean corpuscular volume; MCHC: mean corpuscular hemoglobin concentration; MCH: mean corpuscular hemoglobin.

Effects on serum enzyme levels

The observations of mean ± SE values of biochemical parameters studied from all the treatment groups are presented (Table 7). There was significant (p ≤ 0.05) increase in serum levels of AST, ALP, and albumin and decrease in serum levels of total protein, globulin, glucose, and triglyceride of acrylamide-intoxicated rats as compared to the control group rats. There was no significant difference among serum cholesterol values of the different treatment groups.

Table 7.

Mean values of serum biochemistry of rats exposed to acrylamide for 28 days.^a

Sr. No.	Groups	A	B	C	D
1	AST (IU/L)	162.22 ± 15.17^b	177.57 ± 11.82^b,c	191.59 ± 8.76^b,c	206.94 ± 4.76^c
2	ALT (IU/L)	81.53 ± 8.26	88.32 ± 5.36	95.16 ± 2.87	106.09 ± 11.28
3	ALP (IU/L)	62.21 ± 3.43^b	68.80 ± 6.78^b	73.50 ± 1.27^b,c	84.37 ± 5.52^c
4	Total Protein (g/dL)	8.01 ± 0.24^d	7.56 ± 0.16^c,d	7.12 ± 0.16^b,c	6.56 ± 0.35^b
5	Albumin (g/dL)	3.30 ± 0.18^b	3.84 ± 0.06^c	3.97 ± 0.12^c	4.02 ± 0.21^c
6	Globulin (g/dL)	4.70 ± 0.36^d	3.72 ± 0.21^c	3.14 ± 0.24^b,c	2.54 ± 0.30^b
7	Glucose (mg/dL)	119.25 ± 8.49^d	95.93 ± 5.11^b,c	86.27 ± 3.44^b,c	82.55 ± 2.73^b
8	Creatinine (mg/dL)	0.70 ± 0.07	0.75 ± 0.09	0.70 ± 0.08	0.67 ± 0.06
9	Serum cholesterol (mg/dL)	31.05 ± 2.05	32.79 ± 2.47	32.74 ± 6.34	39.39 ± 3.15
10	Triglyceride (mg/dL)	149.78 ± 9.55^d	133.23 ± 5.94^b,c	125.83 ± 6.22^b,c	118.5 ± 5.99^b

AST: aspartate aminotransferase; ALT: alanine aminotransferases; ALP: alkaline phosphatase.

Histopathological observations by light microscopy

Gross changes in brain and spinal cord were not noticed in any of the groups (Figures 5 to 8). Microscopic examination of liver from control group (A) revealed normal orientation of hepatic parenchyma. Sections of liver from group B revealed vacuolar and granular degenerative changes, which were common finding in liver from this group. Congestion and Kupffer cell proliferation was noticed in liver from two rats of this group. Liver sections from group C showed focal necrosis with Kupffer cell proliferation (Figure 9). Liver sections from high-dose group showed prominent areas of focal necrosis (Figure 10). Severity of degenerative changes was comparatively more than group B and C. The lesions in the kidney from group D were hemorrhages, destruction of tubular epithelium with coagulative necrosis (Figure 11). Degenerative changes observed were mainly restricted to proximal convoluted tubules. Similar but less extensive lesions were observed in group C and mild changes in group B. Focal necrosis and degenerative changes in myocardium was evident in experimental rats that received acrylamide at 20 mg/kg b.w. (Figure 12), while the rats intoxicated with acrylamide at 15 mg/kg b.w. revealed myocardial hemorrhages. The rats treated with acrylamide at 10 mg/kg b.w. did not reveal any pathological lesions. Lungs from control group A did not reveal any pathological lesions. Pronounced edema, emphysema, and round-cell infiltration in the interstitial space was observed in the sections of lungs from the group D. Hemorrhages, congestion, edema, and emphysema were also noticed in group C, but severities of lesions were less as compared to group D. Only congestion was observed in group B.

Brain revealed foamy appearance of neuronal cytoplasm of group B. Neuronal degeneration, separation of nerve fibers, aggregation of glial cells, and necrosis was detected in the sections of brain from group C (Figure 13). Brain sections of group D revealed neuronal degeneration, necrosis edema, severe hemorrhages, and gliosis (Figure 14). Changes were comparatively severe in group D as compared to groups B and C. Sections of spinal cord from group B revealed starting of degenerative process of nerve fibers. Gray matter was relatively intact. Demyelination has also started. The spinal cord lesions from this group were suggestive of poliomalacia. Spinal cord from group C revealed microscopic softening of lumbar cord. Bilateral necrosis with malacia and liquefaction of white matter was evident (Figure 15). Gray matter also revealed loss of myelin. In high-dose group (group D), bilateral demyelination and softening mainly of white matter was observed. There was malacia and liquefaction leading to complete necrosis of white matter of spinal cord (Figure 16). There was no inflammatory reaction and hemorrhages in spinal cord of animals from this treatment group.

Increased MN frequency in acrylamide-intoxicated rats

The observations of genotoxicity (MN assay) mean ± SE values of MN (MN/2000 PCE), PCE per 200 total erythrocytes (PCE/200TE) (Figure 17) and PCE/NCE ratio from all the treatment groups is presented (Table 8).

Table 8.

Mean values of genotoxic parameters studied of rats exposed to acrylamide for 28 days.^a

Group	MN/2000 PCE	PCE/200TE	PCE/NCE ratio
A	1.16 ± 0.47^b	82.33 ± 4.38^b	0.71
B	2.00 ± 0.51^b	91.83 ± 5.71^b	0.78
C	2.66 ± 0.55^b	96.33 ± 5.37^b	0.95
D	5.00 ± 0.96^c	107.16 ± 4.71^c	1.05
E	10.66 ± 1.05^d	88.16 ± 5.17^b	0.98

MN: micronucleus; PCE: polychromatic erythrocytes; NCE: normochromic erythrocytes; ANOVA: analysis of variance.

Antioxidant status

The observations of mean ± SE values of LPO (nanomoles of MDA per gram of tissue), GSH (nanomoles per gram protein), and SOD (units per milligram protein) in liver and kidney from all the treatment groups are presented (Table 9). There was no significant difference among the different treatment groups.

Table 9.

Mean values of oxidative stress status of rats exposed to acrylamide for 28 days.^a

Organ	Groups				Significance
Organ	A	B	C	D	Significance
LPO (nanomoles of MDA per gram of tissue)
Liver	38.20 ± 3.06	42.75 ± 5.27	48.20 ± 4.05	61.53 ± 10.28	NS
Kidney	39.61 ± 7.12	44.48 ± 7.10	49.61 ± 8.49	63.53 ± 2.92	NS
GSH (nanomole per gram protein)
Liver	2.04 ± 0.13	2.62 ± 0.51	2.29 ± 0.37	1.85 ± 0.13	NS
Kidney	1.46 ± 0.09	1.83 ± 0.20	1.90 ± 0.25	1.79 ± 0.24	NS
SOD (units/milligram protein)
Liver	11.91 ± 0.58	13.08 ± 1.12	13.25 ± 1.14	14.16 ± 2.68	NS
Kidney	9.76 ± 1.56	11.40 ± 0.50	11.63 ± 2.20	12.05 ± 1.50	NS

MDA: malondialdehyde; SOD: superoxide dismutase; LPO: lipid peroxidation; NS: non significant; ANOVA: analysis of variance.

Discussion

During the present study, altered toxic manifestations like sitting posture, weakness, splaying of hind limbs, loss of pain sensations, resting on hindlegs, dragging of hind limbs, and incoordinated movements in varying degree in different treatment groups were noticed. The weakness, splaying, dragging of hind limbs, and incoordination of movements encountered in present study were similar to the findings (Yousef and Demerdash, 2006). However, symptoms like tremors, bleeding, spots of blood and cuts on skin along with general hair loss from the body, especially on the face region in mice received acrylamide for 60 days were recorded earlier (Sharma and Jain, 2008).

The increase in latency to move and freezing time was noticed in acrylamide-intoxicated rats during the present study. Decrease in number of squares, extra lines crossed, and rears made by rats during FOB was recorded. Increase in freezing time, decrease in the number of squares crossed, and decrease in the angle of stride in dose-dependent manner was in accordance with the findings made by Youssef and Santi (1997) but differ in length and width of stride from their findings. Increases in inter heel and fourth digits distance in dose-dependent manner was in accordance with the findings made and significant decrease in forelimb and hind limb grip strength observed in the present study were similar to the earlier findings (Youssef and Santi, 1997). Gait abnormalities in present study were in accordance with the earlier findings (Lehning et al., 2002). Manifestation of neurological effects may be due to degeneration observed in spinal cord. Lehning et al. (2002) also indicated that nerve terminal degeneration was the initial neuropathological event in brain stem and spinal cord during acrylamide dosing in rats. The altered FOB finding may also due to modest gene expression changes in the basal ganglia and sensory cortex due to acrylamide toxicity in rats (Bowyer et al., 2009).

The reduction in body weights of rats coincides with tissue damage observed in present study leading to less consumption of feed. The organ weights were reduced, but relative weights were at par with control indicating reduction in absolute organ weight, which is due to reduction in body weights of rats of intoxicated group. Hematological findings of present study were in accordance with the observations (Sharma and Jain, 2008; Miller et al., 1982) whereas differ from the results obtained by Hashimoto et al. (1981). There were no significant changes in TLC, DLC, and hematocrit values. These findings correlated with results obtained by Hashimoto et al. (1981). During the present study, acrylamide reduced TEC and Hb concentration significantly. But, when MCV, MCH, and MCHC values were compared, acrylamide appeared to induce microcytic anemia indicating increase life span of red blood cells in circulation. It might be the result of toxic insult to bone marrow as observed in present study (discussed later).

The increment in serum ALP activity in present experiment in all treatment groups in graded manner was similar to the results reported by Sharma and Jain (2009). The observed elevation in serum AST and ALP values are the outcome of liver damage by acrylamide toxicity in rats. Decrease in serum total protein and globulin due to acrylamide toxicity in rats during the present study was in accordance with the observations made earlier by Yousef and Demerdash (2006). Severe depletion in values of serum triglyceride observed in the study is in accordance with Huang et al. (1982). It coincides with the liver damage observed in the present study. Awad et al. (1998) reported depletion of reduced GSH (reduced CYP concentration) in hepatocytes after acrylamide treatment is responsible for liver damage. We record edema and emphysema in lungs of intoxicated rats which might be outcome of hypostatic congestion in lungs due to less activity of rats in higher intoxicated group. Sections of spleen from all treatment groups including control did not reveal any appreciable pathological findings.

Histopathological examination of brain revealed foamy appearance of cytoplasm, neuronal degeneration, separation of nerve fibers, aggregation of glial cells, and necrosis in the sections of brain. Changes were comparatively severe in group receiving higher dose as compared to groups receiving lower dose. Sections of spinal cord revealed starting of degenerative process of nerve fibers. Gray matter was relatively intact in lower dose groups. In high-dose group, bilateral demyelination and softening mainly of white matter, malacia, and liquefaction leading to complete necrosis of white matter of spinal cord was noticed. There was no inflammatory reaction and hemorrhages in spinal cord of animals from this treatment group. Similar changes in the sections of spinal cord in rats intoxicated with acrylamide were also reported by Lehning et al. (2002). However, Gipon et al. (1977) failed to report any histological abnormalities in the spinal cord of rats receiving acrylamide on alternate days. Ghetti et al. (1973) reported degeneration in spinal cord white matter of rats intoxicated with acrylamide at 20 mg/kg b.w. However, we found that axonal degeneration was not prevalent in brain region or spinal cord of rats exposed to the higher exposure rate. Genotoxic effects of acrylamide were noticed in the present study; it is likely that higher dose level of afferent damage impacts overall functional output of affected nuclei. Consequently, damage to specific nuclei in brain stem and spinal cord might play a significant role in the somatosensory, somatomotor, and autonomic dysfunction that characterizes acrylamide neurotoxicity. The MN and MNPCEs frequency in the present study was observed to be increased in all treatment groups in a graded manner. These findings confirm earlier observations (El-Tohamy and Bayomy, 2008). Glycidamide, a DNA reactive molecule generated in liver would have hampered the erythroblastogenesis in bone marrow (Maniere et al., 2005).

The findings of the present study, that is, values of MDA showed a nonsignificant elevation in liver and kidneys were similar to the findings of Yousef and El Demerdash (2006). We could not draw any conclusion on the parameters studied to know oxidative stress status of acrylamide-intoxicated rats. Sharma and Jain (2008) reported that increase in oxidative stress could be one of the mechanisms of acrylamide toxicity in mice.

Acrylamide has thus influenced almost every aspect of body function and has disorganized the complete physiology of experimental rats. The usage and consumption of acrylamide in our day to day life is further facilitating its entry swift in the food chain affecting every tropic level and thus sustaining a serious threat for human beings. Therefore, an expanded, corollary, and descriptive study at molecular level is needed. Various toxicological effects observed in experimental animals direct us to focus on a deep study and evaluate the possible causes regarding this toxicity of this chemical. It would furnish the scientists with better options, which would help them to search for a median path regarding the use of this chemical and take preventive measures to save the living beings from the hidden disasters of this chemical.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

The present study was supported by grant from Department of Veterinary Pathology; Nagpur Veterinary College, Nagpur, Maharashtra, India.

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