|Year : 2015 | Volume
| Issue : 3 | Page : 765-773
The role of rosemary against acrylamide developmental toxicity on the white matter of the rat spinal cord
Marwa A Elgholam, Abdel-Moneim A Elbarbary, Ahmad S Zolfakar, Hanaa Z Nooh, Abeer E El-Mehi
Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||25-Jan-2015|
|Date of Acceptance||12-Apr-2015|
|Date of Web Publication||22-Oct-2015|
Marwa A Elgholam
Department of Anatomy and Embryology, Faculty of Medicine, Menoufia University, Shebin el kom, 32511 Menoufia
Source of Support: None, Conflict of Interest: None
The aim of this study was to determine the genotoxicity and developmental toxicity of acrylamide (ACR) on the spinal cord white matter of male albino rats and the possible protective role of rosemary.
With increasing number of sources of ACR exposure to the general public, the need for understanding the toxicological risk associated with such exposure is in high demand.
Materials and methods
Eighty male albino rats were used in this study. 20 rats for each group their mothers were given either distilled water, rosemary aqueous extract (RAE) (2.2 ml/kg/twice/week - i.e 0.44 ml/rat) orally, ACR (10 mg/kg/day - i.e 2 mg/rat) orally, or ACR in combination with RAE. The rats were subjected to morphological (clinical) assessent. They were then killed at the age of 1, 7, 14, and 21 days and the thoracic part of the spinal cord was subjected to histological, immunohistochemical, and morphometric studies. Single cell gel electrophoresis (comet assay) was performed on peripheral blood leukocytes.
ACR administration significantly delayed the normal development, reduced the body measurements, and increased the gait score. The spinal cord white matter of ACR-treated rats showed significant reduction in the number of neuroglia, as well as swollen axons and vacuolation. The area percentage of neurofilament and myelin basic protein immunostaining was reduced. Also, ACR led to DNA damage, which was expressed as significant increase in comet tail length, tail DNA%, and tail moment when compared with the control group. ACR toxicity was age dependent. RAE led to improvement in all tested parameters, especially at younger ages.
ACR induced its neurotoxic effect through demyelination of axons and alteration of neurofilament protein content. RAE (Rosmarinus officinalis L.) showed protective effects against developmental toxicity and genotoxicity induced by ACR.
Keywords: Acrylamide, comet assay, development, immunostaining, rosemary aqueous extract, spinal cord, white matter
|How to cite this article:|
Elgholam MA, Elbarbary AMA, Zolfakar AS, Nooh HZ, El-Mehi AE. The role of rosemary against acrylamide developmental toxicity on the white matter of the rat spinal cord. Menoufia Med J 2015;28:765-73
|How to cite this URL:|
Elgholam MA, Elbarbary AMA, Zolfakar AS, Nooh HZ, El-Mehi AE. The role of rosemary against acrylamide developmental toxicity on the white matter of the rat spinal cord. Menoufia Med J [serial online] 2015 [cited 2020 Feb 24];28:765-73. Available from: http://www.mmj.eg.net/text.asp?2015/28/3/765/167899
| Introduction|| |
Acrylamide (ACR) is a white crystalline odorless compound that has multiple chemical and industrial applications . Public health concerns were raised by Swedish studies showing that relatively high levels of ACR were formed during the frying, roasting, or baking of a variety of foods, including potatoes, cereal products, and coffee at temperatures above 120°C . The highest mean daily dietary intake of ACR reported in Alexandria (3.82 μg/kg body weight) was in the age group 3-6 years. Potato chips represent the major source of dietary ACR (46%), followed by fried potato (19%) and finally bread, which contributes to 17% of the mean daily dietary ACR intake .
Neurotoxic effects of ACR have been established in humans and animals . Although much is known about relatively high-dose ACR exposure and its direct effects on the peripheral nervous system, little is known about the developmental effects of ACR toxicity on the central nervous system.
Rosemary (Rosmarinus officinalis) is a herb, composed of dried leaves and flowers, commonly used as spice and flavoring agents in food processing for its desirable flavor . Rosemary leave extract is useful for the treatment and prevention of bronchial asthma, peptic ulcer, inflammatory diseases, hepatotoxicity, and for tension headache, sciatica, low back pain, and renal colic . Carnosic acid (CA), a rosemary phenolic component, has been shown to protect cortical neurons from glutamate and the brain from middle cerebral artery occlusion/reperfusion injury and is used as a protective agent in Parkinson's disease .
| Materials and methods|| |
ACR, a product of Leuconostoc spp., Sigma-Aldrich (St Louis, Missouri, USA), is available in the form of white powder (99% purity). Rosemary is commercially available as green leaves. The air-dried leaves were powdered; then, 10 g of dried plants was dissolved in 100 ml of distilled water and boiled for 5 min. After cooling and passing through filter paper, a clear solution was obtained. The extract was given to the rats over 24 h. Immunomarkers neurofilament (NF; 200 and 68 kDa) and myelin basic protein (MBP) were obtained from Midco Trade Company (Giza, Egypt).
Approval was obtained from the Local Institutional Ethical Committee of Menoufia University for performing the study. Twenty-five sexually mature female albino rats and five male albino rats (for mating) of Sprague-Dawley strain, weighing between 200 and 250 g, obtained from Tanta, Egypt, were used in this study. Five female rats were housed overnight with a sexually mature male albino rat for mating, and every morning vaginal smears were taken and microscopically examined for the presence of sperms. The detection of sperms in the smears was considered as the first day of gestation. The male offspring of these mothers were divided into the following groups.
0The control group consisted of 20 male offspring whose mothers had received distilled water from the sixth day of pregnancy until weaning (postnatal day 21).
This group consisted of 20 male offspring whose mothers had received aqueous extract of rosemary (2.2 ml/kg/twice/week, i.e. 0.44 ml/rat) by oral gavage from the sixth day of pregnancy until postnatal day 21.
This group consisted of 20 male offspring whose mothers had received ACR (10 mg/kg/day, i.e. 2 mg/rat) by oral gavage from the sixth day of pregnancy until postnatal day 21.
This group consisted of 20 male offspring whose mothers had received ACR and aqueous extract of rosemary from the sixth day of pregnancy until postnatal day 21. Five male offspring were killed at ages 1, 7, 14, and 21 days postnatally in each group.
Morphological (clinical) assessment
The offspring were studied and the following were recorded for each group: the time of for appearance, eye opening and ear opening daily, body weight, head length, cervical rump length, and tail length weekly. Behavioral index (Gait score) examination was carried out weekly, and gait scores were examined according to the method of LoPachin and DeCaprio . Body weight was recorded in grams, and head length, head-rump length, and tail length in centimeters; the results were statistically analyzed.
Histological, immunohistochemical, and morphometric studies
The rats were anesthetized and blood samples were collected from the retro-orbital venous plexus. The dorsal aspect of the rats was exposed, and the vertebral column was cut down along its length bilaterally, removing the vertebra. Thereafter, the thoracic part of the spinal cord was removed immediately, fixed in 10% formalin saline for 24 h, and then processed to obtain paraffin blocks. Sections of 4-6 μm thickness were cut using a microtome and stained with hematoxylin and eosin (H&E) and for immunohistochemical staining using MBP and NF. Five different H-stained and E-stained and immunostained sections from five different rats from each group were examined to count the neuroglia in the white matter and the area percentage of brown color of MBP and NF staining using Image analyzer software (image J 1.74v; National Institute of Health, Bethesda, Maryland, USA).
Single cell gel electrophoresis (comet assay)
The comet assay was performed to study DNA damage in peripheral leukocytes as described by Boutet-Robinet et al. .
A volume of 5 μl peripheral blood was added to 120 μl of 0.5% low melting point agarose at 37°C, and then layered onto a precoated microscopic slide with 1.5% normal melting agarose and covered with a coverslip. The agarose was gelled at 4°C, and the coverslip was removed.
The slides were immersed in lysing solution (2.5 mmol/l NaCl, 100 mmol/l EDTA, 10 mmol/l Tris-HCL buffer, pH 10, 1% sodium sarcosinate with 1% Triton X-100 and 10% DMSO) (Sigma-Aldrich) for ~1 h.
Electrophoresis was conducted for 20 min at 25 V and 30 mA. Slides were then stained with ethidium bromide (Sigma-Aldrich) and examined with a Carl Zeiss fluorescent microscope (Zeiss, Jena, Germany) in the Animal Department, Faculty of Science, Menoufia University.
Analysis of results
In damaged cells, breaks appear as fluorescent tails extending from the core toward the anode. Therefore, the migrated nuclear DNA was considered as a damaged DNA spot. The migration was evaluated by measuring the basal nuclear DNA and migrating DNA in 50 randomly selected cells/sample. The data were analyzed using Image J software. The used comet parameters for the evaluation were tail length, tail DNA%, and tail moment, as described by Wojewodzka et al. . These results were subjected to statistical analysis.
Data were expressed as mean ± SD. The results were computed statistically (SPSS for Windows, version 14.0; SPSS Inc., Chicago, Illinois, USA). The nonparametric Kruskal-Wallis test, followed by Tamhane's post-hoc test, was performed. P values greater than 0.05 were considered nonsignificant, whereas P values less than or equal to 0.05 were considered significant and values less than or equal to 0.001 were considered highly significant.
| Results|| |
ACR does not affect female reproduction (females exhibit neurotoxicity). Also, no congenital anomalies were observed in the offspring.
There was no significant (P > 0.05) difference in any of the tested parameters between the control and rosemary-treated group.
Morphological (clinical) assessment of the offspring
ACR-treated rats showed significant (P < 0.05) delay in eye opening, appearance of ear opening, and rat fur compared with the control group, whereas rats that received ACR and rosemary aqueous extract (RAE) showed significantly normal development in terms of eye opening, appearance of ear opening, and rat fur (P < 0.05) when compared with the ACR-treated group [Figure 1].
|Figure 1: Comparison of the developmental milestones between the different experimental groups.|
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The ACR-treated group exhibited gait abnormality from postnatal day 14, which progressed to moderate and severe degrees at postnatal day 21 [Table 1].
There was significant increase in body weight with progression of age (P1 < 0.05). In the ACR-treated group increase in body weight from day 1 to day 21 was significantly lower than that of the control group (P < 0.05). However, supplementation of RAE with ACR led to highly significant increase in body weight with age (P < 0.001) when compared with ACR-treated rats [Table 1].
There was a steady increase in head length, cervical rump length, and tail length with age in the control group. ACR administration showed increase in these parameters, but was significantly lower than that of control, especially at day 21. Administration of RAE with ACR showed significant increase in these parameters with age when compared with the ACR group [Table 1].
Histological, immunohistochemical, and morphometric results
The white matter of the thoracic spinal cord of the control group showed significant (P < 0.05) increase in the number of neuroglia (oligodendroglia and fibrous astrocyte) with progression of age [Figure 2].
|Figure 2: Spinal cord white matter of the control group (a, d, g, and j). The acrylamide-treated group (b, e, h, and k). The rosema ry/acrylamideprotected group (c, f, i, and l). Normal axon profi le (thin arrow), diamond arrow (swollen axon), oligodendroglia (thick arrow), fibrous astrocytes (notched arrow), vacuolation (V) (H&E, ×1000) (m). Histogram showing comparison of the number of neuroglia between the different groups.|
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ACR administration led to significant (P < 0.05) reduction in the number of neurolglia. This reduction was age dependent. Swollen axons and vacuolation were also detected. Vacuolation of the white matter was marked at postnatal days 14 and 21. Addition of RAE with ACR showed increase in the number of neuroglia, which was significant (P < 0.05) at postnatal days 1 and 7, but not significant at days 14 and 21 postnatally. No vacuolation of the white matter was detected. However, a few swollen axons could still be seen [Figure 2].
The control group showed significant (P < 0.05) increase in MBP content in the spinal cord white matter (area percentage of brown color) with age. The ACR-treated group showed significantly (P < 0.05) lower MBP content (area percentage of brown color) when compared with the control group of the same age with more reduction at postnatal day 21. Addition of RAE with ACR showed increased MBP expression (brown color), which was significantly higher when compared with ACR-treated rats [Figure 3].
|Figure 3: MBP immunostaining of the spinal cord white matter of the control group (a, d, g, and j). The acrylamide-treated group (b, e, h and k). The rosemary/acrylamide-protected group (c, f, i, and l) (MBP immunostaining + H&E, ×1000) (m). Histogram showing comparison of the area percentage of MBP immunostaining between the different groups. MBP, myelin basic protein.|
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NF immunostaining of the spinal cord white matter of the control group showed significant (P < 0.05) increase in NF protein content (area percentage of brown color) with age. Rats treated with ACR showed significantly (P < 0.05) lower NF protein content (area percentage of brown color) when compared with the control group of the same age, with more reduction at 21 days. Supplementation of ACR with RAE showed increased NF protein content (area percentage of brown color), which was significantly higher when compared with ACR-treated rats [Figure 4].
|Figure 4: NF immunostaining of the spinal cord white matter of the control group (a, d, g, and j). The acrylamide-treated group (b, e, h, and k). The rosemary/acrylamide-protected group (c, f, i, and l) (NF immunostaining+H&E, ×1000) (m). Histogram showing comparison of the area percentage of NF protein immunostaining between the different groups. NF, neurofilament.|
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Single cell gel electrophoresis (comet assay) results
Rats of the control group showed nonsignificant (P > 0.05) increase in DNA damage with advancement of age, which was expressed as a nonsignificant increase in tail length, %DNA in tail, and tail moment. ACR-treated rats showed significant increase in DNA damage (P < 0.05), which was expressed as significant (P < 0.05) increase in tail length, tail DNA%, and tail moment with age. This damage was more prominent at 14 and 21 days. However, supplementation of rosemary aqueous extract with ACR showed significant (P < 0.05) improvement in DNA by decreasing DNA damage, which was expressed as reduction in tail length, tail DNA%, and tail moment [Table 2] and [Figure 5].
|Figure 5: DNA damage using comet assay in the control group (a, d, g, and j). The acrylamide-treated group (b, e, h, and k). The rosemary/ acrylamide-protected group (c, f, i, and l) (ethidium bromide, × 40).|
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| Discussion|| |
In humans, fetal exposure to ACR through the diet starts in utero, because ACR has been found to cross the placental barrier in vitro . Sorgel et al.  added that ACR passes through the mother's milk to her newborns during lactation.
A constant dose of 10 mg/kg was used because overdoses were found to reduce the reproductive activity of mothers and cause paralysis . ACR did not affect female reproduction as reported by Tyl and Friedman  and no congenital anomalies were reported in the offspring as recorded by El Sayyad et al. .
The fur of the newborns, ear opening, and eye opening appearance were delayed in ACR-treated groups, as reported by Allam et al. . This retardation resulted from impairment of organogenesis, developmental alteration, and growth and protein deficiencies due to malnutrition during development .
Normal pups were not able to walk except at 12-13 days postnatally, as reported by Geisler et al. .
The ACR-treated group exhibited gait abnormality from postnatal day 14, which progressed to moderate and severe degrees at postnatal day 21, as reported by Mehri et al. . Also, LoPachin  found that the gait scores in rats with ACR exposure rose significantly in a time-dependent manner. Lehning et al.  explained this change in gait by moderate to heavy nerve terminal degeneration in many brainstem nuclei and spinal cord gray matter regions.
In the present study, there was significant increase in body weight from day 1 to day 21 postnatally in the control group. However, increase in body weight in the ACR-treated group was significantly lower than that of the control group. Similar results were reported by Allam et al. , and such weight decrease has been proven to be the most sensitive indicator of developmental ACR toxicity . A possible explanation for weight reduction in ACR-treated rats as previously mentioned by Tyl et al.  is that prenatal ACR exposure leads to growth deficiency in the developing fetus. Frieda and William  further explained that ACR affects the function of mammary glands and leads to prolactin reduction, thus impairing lactation. In this study, there was a steady increase in head length, head-rump length, and tail length in the control group, as recorded by Dhungel and Mukerjee . ACR-treated rats showed decrease in these measures when compared with controls. In agreement with these results, El Sayyad et al.  recorded a decrease in crown-rump length of the offspring after ACR supplementation during pregnancy and lactation.
A possible explanation for this growth retardation was given by Sharma and Jain , who said that ACR administration leads to a dose-dependent decrease in T3 and T4 levels, which control protein synthesis and skeletal and mental growth .
This study found that the spinal cord white matter showed significant increase in the number of neuroglia and in the expression of MBP and NF protein with age as reported by Doretto et al.  and Lemke . Likewise, the spinal cord is a NF-enriched tissue; the NF length increases with age and is involved in caliber determination .
The white matter of ACR-treated rats showed swollen axons and vacuolation from axon loss and demyelination, as recorded by Gad-Allah et al. .
LoPachin and Lehning  said that neurofilamentous swelling and eventual degeneration has been traditional morphological features of ACR-induced neuropathy.
ACR treatment showed significant reduction in the number of neuroglia and MBP expression. This reduction was age dependent, as mentioned by McGrath .
Also, Han  found that myelin basic protein (MBP) gene was downregulated in mouse brain by ACR. He added that this downregulation resulted in poor compaction of myelin in nerve fibers, which might be a factor for neurotoxicity of ACR. LoPachin  attributed the significant decrease in myelination to the disturbances of subcellular elements and water distribution in oligodendrocytes and myelin in ACR-treated rats.
Also, this study found that ACR administration led to age-dependent reduction in NF protein content as reported by Tandrup and Jakobsen , who concluded that the only specific alteration in ACR intoxication was the loss of NFs and added that the effect of ACR on the cytoskeleton might be a reduction in the synthesis leading to a decrease in the amount of NFs.
A possible explanation for the axonal atrophy could be that ACR increased phosphorylation of NF proteins and their physiological turnover, which led to cytoskeletal swelling and disruption of axonal transport. Also, ACR activates Ca 2+ -dependent protease in the distal axon, leading to axon degeneration .
ACR administration led to DNA damage, which was expressed by an increase in comet tail length, tail DNA%, and tail moment in peripheral blood leukocytes.
Nixon et al.  observed that ACR administration led to significant dose-dependent increase in DNA damage in germ cells. The mechanisms by which ACR exerts genotoxicity and DNA damage mostly relate to its conjugation to glutathione, leading to depletion of glutathione, which is essential for the deactivation of reactive oxygen species that can damage DNA . Also, ACR and its metabolite glycidamide can cause chromosomal damage . Alternatively, glycidamide may also induce oxidative stress, which can lead to the production of free radicals and reactive oxygen species .
In the present work, RAE (R. officinalis L.) was used as an antioxidant as it is being increasingly used as a food additive, as recorded by El-Beltagi and Badawi .
In the present study, supplementation of RAE led to improvement in developmental milestones, in gait score, and on clinical, histopathological, morphometric, immunohistochemical, and genetic assessment. This can be explained by Shimojo et al. , who revealed that rosemary extract had the potency to decrease the gait score and significantly improve motor performance of mice. They added that rosemary treatment suppressed body weight reduction of human superoxide dismutase (SOD1) transgenic mice.
Moreover, Elomri et al.  concluded that the extract of rosemary and of two pure compounds rosmarinic acid and CA promoted the neurite outgrowth and cell differentiation of the neural cell model of pheochromocytoma and enhanced the acetylcholinesterase activity in the same way as the nerve growth factor.
Park  found that isorosmanol, isolated from rosemary, significantly increased the immunoreactivities of phosphorylated NF-H and NF-M, and total NF-H. He added that isorosmanol induces neurotrophic outgrowth by increasing the levels of NF proteins.
This improvement was explained by Paxinos , who found that carnosoic acid is involved in the synthesis of nerve growth factor, which is necessary for the growth and maintenance of nerve tissue, which stimulates the growth of new oligodendrocytes. Also, supplementation of RAE protected against the increase in levels of oxidative stress markers .
Similarly, Perez-Sanchez et al.  reported that combination of rosemary and citrus extracts can decrease the number of ultraviolet-induced DNA breaks using the comet assay and hence either attenuates the genotoxic effects of ultraviolet radiation or protects the DNA repair machinery. Moreover, Packer et al.  said that the antigenotoxic effects of rosemary components mediated by their antioxidant properties were responsible for scavenging free radicals involved in DNA oxidation. Indeed, CA, carnosol, rosmarinic acid, and ursolic acid effectively inhibited DNA strand breakage, suggesting effective scavenging of the hydroxyl radicals .
| Conclusion|| |
ACR induced growth and developmental retardation in newborn rats when their mothers were exposed to it during the gestation and lactation periods. ACR induced its neurotoxic effect through demyelination of axons and alteration of NF protein content. It also exerted genotoxic effects that led to the destruction of DNA. RAE (R. officinalis L.) showed protective effects against ACR-induced developmental neurotoxicity and genotoxicity.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]