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Year : 2021  |  Volume : 9  |  Issue : 3  |  Page : 133-143

Melatonin and Vitamin C modulate cassava diet-induced alteration in reproductive and thyroid functions

1 Department of Physiology, College of Health Sciences, University of Ilorin, Ilorin, Kwara, Nigeria
2 Department of Physiology, School of Medicine and Pharmacy, College of Medicine and Health Sciences, University of Rwanda, Huye, Republic of Rwanda

Date of Submission04-Apr-2021
Date of Decision01-Jun-2021
Date of Acceptance02-Jun-2021
Date of Web Publication30-Nov-2021

Correspondence Address:
Mr. Oloruntobi Oluwasegun Maliki
Department of Physiology, College of Health Sciences, University of Ilorin, Ilorin, Kwara
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/njecp.njecp_9_21

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Background: Cyanide, present in cassava, causes adverse effects on the thyroid and male reproductive functions and its poisoning generates free radical and oxidative stress. Melatonin and Vitamin C are antioxidants that improve conditions associated with oxidative stress. Aims and objectives: We evaluated the effects of melatonin and/ or Vitamin C on body weight, thyroid functions, and reproductive parameters in cyanide-enriched cassava-fortified diet (CD)-treated rats and their possible mechanisms of actions. Materials and Methods: Thirty male rats were divided into six groups (n = 5 each): Group I – Control, Group II – Melatonin, Group III – Vitamin C, Group IV – CD, Group V – CD + Melatonin, and Group VI – CD + Melatonin + Vitamin C. The control received normal saline, while melatonin and Vitamin C groups were dosed orally at 15 mg/kg melatonin and 100 mg/kg Vitamin C, respectively, CD group was fed with 40% cassava-fortified diet only, while other groups received the combination of the treatments. Results: In CD-treated rats, the sperm parameters were not affected but sperm count was insignificantly increased by melatonin, while melatonin + Vitamin C significantly increased all semen parameters. Neither CD only nor co-administration with melatonin and/or Vitamin C affected plasma luteinizing hormone and testosterone. The CD increased triiodothyronine (T3), but the increase was abolished by melatonin. Moreover, the CD increased thyroxine (T4), which was neither affected by melatonin alone nor its combination with Vitamin C. The levels of thyroid-stimulating hormone were not different across all treatment groups. The CD increased the thiocyanate, which was ameliorated by melatonin but abolished by combination of melatonin and Vitamin C. The CD also decreased the total antioxidant capacity level, which was abolished by melatonin. The CD increased weight gain, thyroid hormone, and oxidative stress but had no effect on semen parameters and reproductive hormones. Conclusion: Melatonin and Vitamin C attenuate the effects of CD on weight, thyroid hormones, and oxidative stress.

Keywords: Cassava, cyanide, sperm, thiocyanate, thyroid, total antioxidant capacity

How to cite this article:
Maliki OO, Alagbonsi AI, Ibitoye CM, Olayaki LA. Melatonin and Vitamin C modulate cassava diet-induced alteration in reproductive and thyroid functions. Niger J Exp Clin Biosci 2021;9:133-43

How to cite this URL:
Maliki OO, Alagbonsi AI, Ibitoye CM, Olayaki LA. Melatonin and Vitamin C modulate cassava diet-induced alteration in reproductive and thyroid functions. Niger J Exp Clin Biosci [serial online] 2021 [cited 2022 Aug 14];9:133-43. Available from: https://www.njecbonline.org/text.asp?2021/9/3/133/331560

  Introduction Top

Cassava (Manihot spp.) has been a staple crop of the tropics for many years. Owing to the presence of cyanogenic glycosides in cassava, various methods of detoxification have been employed.[1],[2] The traditional method of reducing cassava toxicity in Nigeria is by fermentation. Products from such fermentation include garri flour and fufu.[3] Garri flour derived from cassava is a major staple food for many people in most African and Latin American countries. The soaking of cassava in water, rinsing and baking effectively reduce cassava cyanide content, but improper processing techniques can yield toxic food product.[3] The efficiency of cyanogen removal depends largely on the kinds of unit operations involved in the processing method as well as the initial cyanogen load.[3] The toxicity of cyanogenic glycosides results from the production of hydrogen cyanide and consequently cyanide poisoning.

Cyanide is a highly toxic compound with both acute and chronic effects[4] stemming from ability to inhibit respiration and the action on some metalloenzymes.[5] Large percentages of the population are exposed to very low levels of cyanide in the general environment. There are, however, specific subgroups with higher potential for exposure. These include individuals involved in large-scale processing of cassava and those consuming significant quantities of improperly prepared foods containing cyanogen glycosides.[6] The cassava root contains a sufficient amount of cyanogens which require special processing to reduce the danger of toxicity. Cyanide has been shown to be a reproductive toxicant in male dog.[7] Manzano et al.[8] examined the effects of subchronic potassium cyanide in large white pigs and observed histological alterations of the thyroid gland in all the treated animals. Okafor and Onyema[9] also reported increase in the serum level of liver enzymes of rats fed with the varied proportion of unprocessed cassava. Paulinus and Obaika[10] observed the toxic effect of prolonged intake of cassava-borne organic cyanide and inorganic cyanide in some rabbit tissues. There was increase in the level of serum lactate dehydrogenase following cyanide exposure which is an indication of shift in aerobic to anaerobic metabolism, causing lactic acidosis.[11]

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine of molecular weight 232 g/mol, which is synthesized from the essential amino acid, tryptophan, through serotonin.[12] Melatonin was first isolated in 1958 as a neurohormone mainly synthesized and secreted from the pineal gland.[13] Since its discovery, further investigations have revealed that it is also produced by several other organs. It has been found in the gastrointestinal tract,[14] brain,[15] eye,[16] lungs,[17] kidney,[18] liver,[19] thyroid, thymus, pancreas,[20] immune system,[21] and reproductive system.[22] Melatonin helps to regulate circadian rhythm and the sleep-wake cycle,[23] acts as oxygen and other reactive oxygen species (ROS) scavenger during the production of free radicals in metabolic processes,[24] and serves to boost sperm[25] and oocyte[26] qualities, among others. Binding sites of melatonin have been detected in the reproductive system of different species,[27],[28] hence, it is assumable that melatonin exerts its actions through direct interaction with the steroidogenic cells of the reproductive organs.[29] Similarly, Vitamin C (ascorbic acid) is a water-soluble vitamin obtained from human's diet only. Ascorbic acid reduces tocopheryl radical formed by the reaction of Vitamin E with lipid radicals, protects membranes against oxidation, prevents lipid peroxidation, and affects the regeneration of Vitamin E.[30]

The exact mechanism by which cyanide exerts a damaging action on tissues is not clear. However, some researchers have proposed that oxidative stress may be implicated in the harmful effects of cyanide poisoning,[31] by increasing ROS and reactive nitrogen species (RNS)[32] and inhibiting antioxidant systems and mitochondrial function. The ameliorative effects of antioxidants on toxicities elicited by many toxicants have also been reported. For instance, Obianime and Roberts[33] have observed the ameliorative effect of Vitamin C on kidney and testes in cadmium-induced toxicity of male Wistar rats. Vitamin C has also been shown to ameliorate testicular toxicity due to lead exposure in albino rats[34] and associated with improvement in semen quality in humans,[35] rabbit,[36] and rats.[37] Melatonin and Vitamin C have been reported to ameliorate cannabis-induced gonadotoxicity in male rats in vivo and in vitro.[38],[39],[40] The inhibitory effect of thiocyanate exposure on thyroid growth and function has also been reported.[41]

There are several data on the effect of melatonin on the adverse effects caused by inorganic cyanide, but only few studies have reported the effect of melatonin on the organic cyanide from natural food products (e.g., cassava, apricot seeds, and choke cherries). 6-hydroxymelatonin, a major metabolite of melatonin, has been observed to significantly reduce the potassium cyanide (KCN)-induced superoxide anions production and lipid peroxidase content and exert the similar neuroprotective effects against KCN-induced neurotoxicity in rat brain.[42] Since cyanide is known to have toxic effect on the thyroid and reproductive glands, dysfunction of these glands in chronic consumers of cyanide-enriched cassava diet (CD) is a likely concern that needs careful investigation. Moreover, the possible ameliorative effect of antioxidant supplements such as melatonin and Vitamin C on thyroid and reproductive toxicities in CD consumers needs to be explored. The present study aimed at investigating the effect of melatonin and Vitamin C on body weight, thyroid function, and gonadal parameters in male rats treated with CD and their possible mechanisms of actions.

  Materials and Methods Top


Thirty adult male Wistar rats (weight range: 180–220 g) were obtained from a trusted commercial breeder. They were housed in wooden cages maintained under standard conditions (12-h light/dark cycle, 27°C–30°C, 50%–80% relative humidity) and were acclimatized in the laboratory for 2 weeks before the commencement of the study. The rats were fed with standard palletized rodent diet (Ace Feeds, Ibadan, Nigeria) and water ad libitum. All the animals were well catered according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Science and approved by the Ethical Research Committee of the University of Ilorin, Nigeria.

Experimental protocol

Freshly harvested cassava was obtained from International Institute of Tropical Agriculture, Ibadan, Nigeria after proper identification by Mr. Peter Iluebbey (International Trial Manager, Yam-barn Unit) of the Institute, and the specie identification number (TM-91934) was provided by the Institute. The cassava root was peeled to remove the external coat (brownish part), and whitish part was sliced into small pieces. It was later air-dried and pounded into the size of the grower's feed. This enables the cassava to mix properly with grower's feed according to the required proportion. Sixty per cent grower's feed was mixed with 40% cassava root meal to formulate the cassava (CD) used in this study; this form of inadequately processed cassava (CD) was used throughout the study.

Following an established method for the determination of animal sample size,[43] the 30 animals were blindly divided (allocation to groups was done by an invited neutral person who knew nothing about the study) into six treatment groups (n = 5 each) after acclimatization as follows: Group I – Control, Group II – Melatonin, Group III – Vitamin C, Group IV – CD, Group V – CD + Melatonin, and Group VI – CD + Melatonin + Vitamin C. The control received normal saline, while melatonin and Vitamin C groups were dosed orally at 15 mg/kg and 100 mg/kg body weight, CD group was fed with 40% CD only, while Group V received melatonin and VI received melatonin and Vitamin C in addition to CD. Except in the groups that received CD, other animal groups received standard animal diet. All animals had unrestricted access to their assigned diet throughout the experimental period, while melatonin and Vitamin C were administered daily to the animals in the same order between 10:00 am and 12:00 noon for 28 days.

Animals were euthanized a day after the last treatment under chloroform anesthesia and blood was collected by cardiac puncture after dissection. The blood was then spun for 10 min at 4000 revolutions per minute and the supernatant plasma from each centrifuged blood was transferred into separate plain bottles and stored at 20°C before assays of the biochemical parameters.

Determination of Biochemical parameters

Biochemical parameters, including total antioxidant capacity (TAC), luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), thyroid-stimulating hormone (TSH), triiodothyronine (T3), Thyroxine (T4), and serum thiocyanate, were estimated in this study.

The TAC (Product Code: BXC0553; Fortress diagnostics Limited, United Kingdom), LH (Catalog No: BXE0651A; Fortress diagnostics Limited, United Kingdom), FSH (Catalog No: BXE0631A; Fortress diagnostics Limited, United Kingdom), testosterone (Catalog No: TE187S; Calbiotech Inc., Spring Valley), TSH (Calbiotech, Spring Valley, CA), T3 (Calbiotech, Spring Valley, CA), and T4 (Calbiotech, Spring Valley, CA) were determined spectrophotometrically (Spectramax Plus; Molecular Devices, Sunnyvale, CA, USA) according to the kit manufacturers' instructions. The serum thiocyanate was determined as previously described.[44]

For the assay of T3, 50 μl of the control, specimen, and serum reference were added into the designated well, followed by 100 μl of T3-enzyme conjugate solution and gentle swirling for 20–30 s. For the assay of T4, 25 μl of the standards, specimen, and control were added into the designated well, followed by the addition of 50 μl each of the working T4-enzyme conjugates solution and T4-antibody-biotin solution to all wells. For assay of TSH, 50 μl of TSH standards, sample, and control were added into the designated wells and 100 μl of the conjugate reagent was added. In all the assays, the microplate was then incubated for 60 min. The wells were emptied of the liquid and were washed three times with wash buffer, followed by blotting on absorbent paper towels. Then, 100 μl of 3,3', 5, 5'-Tetramethylbenzidine substrate solution was added to all wells, and the plates were then covered and incubated for 15 min, followed by 50 μl of stop solution and gentle mixing for 15–20 s. Within 15 min of adding stop solution, the ELISA reading of the absorbance was done at 450 nm. The concentration of T3, T4, or TSH was extrapolated from the standard curve plotted for each of them.

Determination of cyanogenic glycoside

The method used for this assay was obtained from the Association of Official Analytic Chemist.[45] Briefly, 4 g of sample was soaked in a mixture containing 40 ml of distilled water and 2 ml of orthophosphoric acid. The mixture was totally steroid stopped and left overnight at room temperature to set free all bounded hydrocyanide acid. The resulting sample was transferred into the distillation flask and a drop of paraffin wax was added (anti-foam 1 mg agent) together with broken chips (antibump).

The distillation flask was filled to other distillation apparatus and then distilled. About 5 ml of distillate was then collected in the receiving flask that contain 0.1 g of sodium hydroxide pellets. The distillate was then transferred to 50 ml volumetric flask and made up to mark with distilled water. It was collected and placed in the conical flask after which 1.6 ml of 5% potassium iodide was added to the filtrate. The resulting mixture was titrated against 0.01. The calculated cyanide content in the cassava used for this study from the method stated above was 3.71 mg/kg.

Estimation of epididymal semen parameters

Sperm motility, estimated as the percentage of sperm that manifests progressive motility, was determined as previously described.[46],[47] Briefly, the sperm suspension was diluted in 1 ml of normal saline solution. About 10 L was pipetted onto a clean grease-free glass slide. A cover slip was lowered onto the sample on the slide, avoiding air bubbles, and the slide was examined using a microscope with a 40× objective. At least, six widely spaced fields were examined to provide an estimate of the percentage of the progressively motile sperm cells. The sperm cells with progressive motility were estimated and recorded as (N) while the total number of all the sperm cells counted was recorded as (T). Sperm motility (%) was calculated using (N/T × 100%).

Percentage of morphologically normal sperm was estimated by the method previously described.[46],[48] The principle is based on the ability of morphologically normal sperm to appear white in color as the plasma membrane will prevent the dye to enter, while abnormal sperms take up the dye and stain dark color. The microscope slides and the eosin stain were prewarmed to room temperature. One milliliter of the sperm suspension – normal saline solution was transferred to a test tube and 2 drops of 1% eosin were added and mixed gently for agitation. This was incubated for 45–60 min to allow its proper staining and then resuspended with a Pasteur pipette. A clean grease-free glass slide was used. Potential damage to the sperm cells was avoided. One or 2 drops of the stained sperm were placed approximately 1 cm from the end of the slide lying on a flat surface. A second slide was held with the slide's long edge gently touching across the width of the sperm slide and pulled across to produce a sperm smear. After air-drying the slide, using a microscope at ×100, the sperm cells were examined. The sperm along the periphery was normally excluded from the examination because there is a greater tendency for artifacts to occur in these regions. At least, five fields were viewed covering the whole slide. Examples of morphological abnormalities are double-headed, elongated head, pyriform head, bent head, bent tail, bent mid-piece, coiled tail, double tail, headless, tailless, etc. All those with normal morphology were recorded as N, while the total number of the counted spermatozoa was recorded as T. The percentage sperm morphology was calculated as N/T × 100%.

The testes from each rat were carefully exposed and one of them was removed together with its epididymis. For each separated epididymis, the caudal part was removed and placed in a beaker containing 1 ml of normal saline solution. It was macerated with a pair of sharp scissors and left for few minutes to liberate the sperm cells into the normal saline. Semen drops were placed on a clean grease-free glass slide and two drops of warm 2.9% sodium citrate were added. The improved Neubauer counting chamber was charged with the semen solution and the number of sperm cells, appearing as black dots, was counted in 25 small squares within the central counting area of the counting chamber as earlier described.[49],[50] Using the ×100 with the condenser iris closed sufficiently to give good contrast, the number of spermatozoa was counted in an area of 2 mm2, i.e., 2 large squares.[49]

Sperm viability was assessed by eosin-nigrosin assay as previously described.[51] The percentage of viable sperm (sperm head unstained indicating living sperm) and nonviable sperm (sperm head stained indicating dead spermatozoa) was assessed by counting a minimum of 100 spermatozoa. Replicate counts of 100 sperm on each of two slides were performed. These were then repeated if >5% difference was found.

Statistical analysis

Data were blindly analyzed (by an independent scientist) using the Statistical Package for the Social Sciences (SPSS) software version 16 (IBM Corporation, Armonk, NY, USA) and expressed as means ± standard error of the mean of the values. Two-way analysis of variance was used to compare the data, followed by post hoc least significant difference multiple comparison test to determine the significance at P < 0.05. In addition to the comparison of all groups with the control (normal saline) group, the groups that received CD in combination with melatonin and/or Vitamin C were additionally compared to the group that received CD only.

  Results Top

Proximate composition of cassava

The proximate composition of the cassava sample is described in terms of nutrients and their respective percentage of composition. It shows that the cassava sample has dry matter as its highest composition, followed by moisture, crude fibres, crude proteins, total ash, and total fat [Table 1].
Table 1: Proximate composition of the cassava

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Bodyweight in rats given cassava with(out) melatonin and/or Vitamin C

When compared to the baseline, there were weekly increases in the weight of the rats in all the groups. There was no statistical difference in the baseline weight of the rats across various groups but the weight increased in all the experimental groups during the observation period. At week 1, there was an increase in the weight of rats that received melatonin (214.4 ± 6.0 g), CD (185.2 ± 22.9 g), CD + Melatonin (208.4 ± 5.5 g), Vitamin C (190.8 ± 14.8 g), and CD + Melatonin + Vitamin C (186.0 ± 9.2 g). Similarly, at week 2, there was an increase in the weight of rats that received melatonin (215.0 ± 5.7 g), CD (180.0 ± 20.5 g), CD + melatonin (188.0 ± 21.4 g), Vitamin C (192.0 ± 11.6 g), and CD + melatonin + Vitamin C (190.0 ± 8.4 g). At week 3 also, there was an increase in the weight of rats that received melatonin (212.0 ± 3.7 g), CD (181.0 ± 20.6 g), CD + melatonin (183.0 ± 22.9 g), Vitamin C (195.0 ± 11.4 g), and CD + Melatonin + Vitamin C (187.0 ± 11.6 g). Finally, at week 4, the weight increased in rats that received melatonin (212.0 ± 5.2 g), CD (186.0 ± 21.4 g), Vitamin C (205.0 ± 11.5 g), and CD + Melatonin + Vitamin C (205.0 ± 7.8 g) [Figure 1]a.
Figure 1: Effect of melatonin and/or Vitamin C on bodyweight (a) and feed intake (b) of rats treated with cassava-diet. CD: Cyanide-enriched cassava-diet; Mel: Melatonin; Vit C: Vitamin C

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Feed intake in rats given cassava with(out) melatonin and/or Vitamin C

Before administration, there was no statistical difference in the feed intake of the rats across various groups. At week 1, the feed intake increased in rats that received CD (35.0 ± 1.6 g), reduced in rats that received melatonin (20.0 ± 1.6 g), but unchanged in other groups. At week 2, the feed intake increased in rats that received melatonin (35.0 ± 1.4 g) and CD + melatonin + Vitamin C (40.0 ± 2.7 g), reduced in rats that received CD + Melatonin (15.0 ± 1.6 g), but unchanged in other groups. At week 3 also, the feed intake decreased in all groups (except CD). Finally, at week 4, the feed intake increased in rats that received CD (25.0 ± 3.5 g) and Vitamin C (25.0 ± 1.6 g) but decreased in rats that received melatonin (15.0 ± 1.6 g) and CD + Melatonin + Vitamin C (11.0 ± 1.0 g) [Figure 1]b.

Sperm count and concentration in rats given cyanide-enriched cassava-diet with(out) melatonin and/or Vitamin C

Vitamin C, but not melatonin, increased the semen parameters (except sperm viability) in rats when compared to control. The CD did not affect the semen parameters, while the sperm count, but not other semen parameters, was increased by melatonin in CD-treated rats. Combination of melatonin and Vitamin C in CD-treated rats increased the semen parameters when compared to control and CD only [Table 2].
Table 2: Effect of melatonin and/or Vitamin C on semen parameters of rats treated with cyanide-enriched cassava-fortified diet

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Serum follicle-stimulating hormone, luteinising hormone, and thyroid-stimulating hormone, levels in rats given cyanide-enriched cassava-diet with(out) melatonin and/or Vitamin C

The insignificant decrease in plasma FSH caused by CD was potentiated by melatonin coadministration to a noticeable level. However, neither CD alone nor its coadministration with melatonin and/or Vitamin C caused any noticeable effect on the plasma LH and testosterone when compared to the control [Table 3]. The levels of TSH were not different across all treatment groups [Table 3].
Table 3: Effect of melatonin and/or Vitamin C on gonadotropins and testosterone of rats treated with cyanide-enriched cassava-fortified diet

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Thyroid hormones in rats given cyanide-enriched cassava-diet with(out) melatonin and/or Vitamin C

Melatonin (0.76 ± 0.08 ng/ml) or Vitamin C (0.61 ± 0.11 ng/ml) caused no significant change in the plasma T3 when compared to control (0.68 ± 0.1 ng/ml). The CD (1.11 ± 0.12 ng/ml) increased T3 but the increase was abolished by melatonin only (0.69 ± 0.13 ng/ml) [Figure 2]a.
Figure 2: Effect of melatonin and/or Vitamin C on triiodothyronine (a) and thyroxine (b) levels of rats treated with cassava-diet. *P < 0.05 when compared to control; #P < 0.05 when compared to cassava-diet; Mel: Melatonin; Vit C: Vitamin C

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Melatonin (0.98 ± 0.4 μg/ml) or Vitamin C (1.21 ± 0.5 μg/ml) did not affect T4 when compared to control (0.63 ± 0.1 μg/ml). The CD (1.21 ± 0.11 μg/ml) increased T4 which was neither affected by melatonin alone (1.15 ± 0.13 μg/ml) nor its combination with Vitamin C (1.35 ± 0.12 μg/ml) [Figure 2]b.

Serum thiocyanate and total antioxidant capacity in rats given cyanide-enriched cassava-diet with(out) melatonin and/or Vitamin C

Vitamin C (4.95 ± 0.12 μg/ml), but not melatonin (4.16 ± 0.99), increased the thiocyanate level when compared to control (4.19 ± 0.37 μg/ml). The CD (7.94 ± 0.61 μg/ml) increased the thiocyanate level, which was ameliorated by melatonin (5.29 ± 0.31 μg/ml) but abolished by combination of melatonin and Vitamin C (4.29 ± 0.59 μg/ml) [Figure 3].
Figure 3: Effect of melatonin and/or Vitamin C on thiocyanate of rats treated with cassava-diet. *P < 0.05 when compared to control; #P < 0.05 when compared to cassava-diet; Mel: Melatonin; Vit C: Vitamin C

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Melatonin (1.30 ± 0.35 μg/ml) or Vitamin C (1.96 ± 0.08 μg/ml) did not affect TAC when compared to control (1.90 ± 0.09 μg/ml). The CD (1.69 ± 0.03 μg/ml) decreased the TAC level, which was abolished by melatonin only (1.85 ± 0.06 μg/ml) [Figure 4].
Figure 4: Effect of melatonin and/or Vitamin C on total antioxidant capacity of rats treated with cassava-diet. *P < 0.05 when compared to control; #P < 0.05 when compared to cassava-diet; Mel: Melatonin; Vit C: Vitamin C

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  Discussion Top

The increase in feed intake in CD group suggests that the cassava inclusion in the diet had a positive effect on the animal which in turn improved their weight gain.[52] The previous study had also observed that cassava inclusion can increase feed intake and consequently the weight of treated animals.[53] The high nutritional value of cassava has been reported, having 112 calories per 100 g compared to sweet potatoes and beets having 76 calories per 100 g and 44 calories per 100 g, respectively.[54] Its ability to provide high calories has made it an important crop for developing countries. Consumption of high calories diet has been associated with weight gain and obesity. For instance, dietary energy density was associated with higher body mass index, waist circumference, elevated fasting insulin, and metabolic syndrome in US adults.[55] Normal-weight persons have also been shown to have diets with a lower energy density than obese persons.[56] The increase in weight gain by CD in the present study could also be partly associated to its high calories as previously reported. While the discrepancy in our observation of high weight gain and the reduction in weight gain reported by others[57],[58],[59] cannot be convincingly justified at the moment, we speculate that the dosage of the cassava administered, species of animals, the geographical effect on different cassava samples, and processing methods could be culpable.

Consumption of foods with low energy density (kcal/g) has been said to reduce energy intake and has been recommended for weight management. For instance, men and women with low-energy-dense diet had a lower energy intake (approximately 425 and 275 kcal/day less) than did those with a high-energy dense diet, even though they consumed more food (approximately 400 and 300 g/day more, respectively). Moreover, persons with high fruits and vegetable intake had the lowest energy density values and the lowest obesity prevalence.[56] In this study, we investigated if supplementation of CD with antioxidants having negligible energy density will increase feed consumption and also reduce the bodyweight of rats as noted in humans by Ledikwe et al.[56] and Mendoza et al.[55] We observed an increase in body weights of rats that received melatonin and Vitamin C supplements in addition to CD throughout the experimental period. We also found that the feed intake was higher in rats that received cassava but lower or unchanged in rats that received melatonin and/or Vitamin C with (out) cassava at weeks 1 and 4. Contrarily, the feed intake was higher in rats that received melatonin and Vitamin C supplements in addition to cassava at week 2 and also higher in all groups (except in those that received CD) at week 3. Thus, our study suggests that the effect of energy density on weight gain and feed consumption are time-dependent.

Healthy individuals have a small amount of cyanide in their bodies as concentration up to 50 μg/100 g tissue and 106 μg/L have been found in different organs and plasma, respectively.[60] Levels of cyanide metabolite, thiocyanate, in blood serum, plasma, and urine have been employed as indicators of high cyanide exposure in humans, while variations have been noted at low levels.[61] Thiocyanate, the major detoxification product of cyanide, prevents the uptake of iodine and acts as a goitrogenic agent. This effect could be more pronounced in individual with decreased capacity to excrete thiocyanate due to kidney dysfunction.[62] Moreover, long-term consumption of inadequately-prepared cassava may lead to reproductive dysfunction and consequently infertility.

Is the cassava-induced increase in body weight related to alteration in thyroid functions? It has been reported that cyanide causes a reduction in the growth rate of hens by inhibiting intrathyroidal-stimulating hormone and thereby causing a reduction in thyroxine level which is necessary for growth.[63] In a Mozambique rural population affected by spastic paraparesis, the antithyroid effect of thiocyanate from cassava-derived cyanide exposure manifested as decrease in serum T4 but increase in serum T3, T3/T4 ratio, and TSH.[64] Thiocyanate also inhibited sodium-iodide symporter, thus reducing the transport of iodine from circulation into thyroid follicular cells, which will impair thyroid hormone synthesis.[41] In weaned mice, thiocyanate decreased thyroid T3, T4, and iodine contents but increased plasma TSH with corresponding hyperthrophy of the thyroid gland, all of which followed recovery after thiocyanate withdrawal.[64] Fresh cassava root-induced elevation of serum thiocyanate was accompanied with no change in thyroid gland size and thus no goiter.[66] However, our own study disagrees with these previous studies and showed hyperthyroid effect of CD, as we noticed increase in the plasma T3 and T4 without any corresponding change in the TSH. Our study partly agrees with that of Daniel et al.[67] that also noticed cyanide-induced hyperthyroidism but slightly disagrees with their study as our own hyperthyroidism is independent on thyrotropin like theirs. Daniel et al.[67] treated male Wistar rats with hexacyanoferrate III solution for 56 days and reported significant increase in the levels of thyroid hormones (T3 and T4) but reduction in TSH, while the thyroid gland showed marked epithelial hyperplasia with cellular degeneration and scanty cytoplasm. We therefore speculate that the increase in weight gain elicited by CD in our study is related to hyperthyroidism-induced increase in feed intake in the rats.

Does cassava and its cyanide cause oxidative stress? Serum thiocyanate is a stable metabolite and a useful biomarker of cyanide exposure,[64] whose implications in oxidative stress have been well documented.[44] For instance, prolonged sublethal cyanide administration caused a decline in superoxide dismutase activity in red blood cells and catalase activities in some tissues of cyanide-toxified rats.[67],[68],[69] Cyanide intoxication has been linked to increasing lipid peroxidation, leading to the production of malondialdehyde (MDA) which is a pro-oxidant that causes oxidative stress.[70],[71] Hariharakrishnan et al.[72] also reported that various concentrations of cyanide caused cytotoxicity in Rhesus monkey kidney epithelial cells, which was accompanied by the elevation of MDA, ROS, RNS, and diminished cellular antioxidant status (reduced glutathione, glutathione peroxidase, superoxide dismutase, and catalase). Increase in serum aminotransferases (aspartate and alanine aminotransferase) have also been observed following cyanide exposure, indicating damage to the cell membrane of the liver.[73] In the present study, we observed that CD increased thiocyanate and reduced the TAC. These observations suggest that CD has pro-oxidant effect and is in agreement with previous studies cited in this paragraph.

Is there a link between the cassava-induced hyperthyroidism and oxidative stress? The involvement of ROS and oxidative stress in the development of hyperthyroidism and autoimmune diseases such as Graves' disease has been well documented. For instance, hyperthyroidism increases oxygen consumption, dysfunction in the mitochondrial respiratory chain, elevated intracellular adenosine triphosphate consumption, and increased ROS production.[74],[75] Genesis of Graves' disease and its orbitopathy[76] in addition to hyperthyroidism-induced damage such as thyrotoxic myopathy and cardiomyopathy[77] have been strongly linked to oxidative stress. Untreated hyperthyroidism also reportedly increased oxidative stress parameters, while restoration of euthyroidism with antithyroid drug reversed the biochemical abnormalities associated with oxidative stress.[78] In fact, animal and human studies suggest that increased ROS directly contributes to some clinical manifestations of the disease. Our simultaneous observation of hyperthyroidism and oxidative stress in cassava-treated rats shows that there is a link between these two conditions as reported by others.

Can the oxidative stress be ameliorated by melatonin and/or Vitamin C? Treatment of 24 hyperthyroid patients with propylthiouracil for 5 days combined with Vitamin C for 1 month potentiated the antioxidant defense system and oxidative stress in them.[79] Antioxidants treatment also improved clinical picture of hyperthyroid patients and led to earlier attainment of euthyroid state.[80],[81] Asayama et al.[82] also reported that Vitamin E protects against thyroxine-induced lipid peroxidation in muscles. Similarly, we also observed in this study that melatonin and/or Vitamin C ameliorate cassava-induced oxidative stress and hyperthyroidism in rats.

Exposure of dogs to cassava-borne cyanide diet for 14 weeks has been reported to elicit testicular degenerative changes and liver lesion.[7] We have reported in our previous in vivo study that melatonin and/or Vitamin C increased sperm motility and morphology in rat by enhancing the antioxidant scavenging process measured with the ROS-TAC score.[40] Moreover, we reported that melatonin and Vitamin C ameliorate Cannabis sativa-induced spermatotoxicity when combined but exacerbates it when administered separately in rats in vivo by altering the endocrine and redox mechanisms.[38],[39],[40] We also reported that melatonin increases rats' sperm motility and kinematics in vitro, and that it ameliorates the reductions in sperm motility and kinematics elicited by tetrahydrocannabinol (the psychoactive substance in C. sativa).[83] In the present study, we investigated whether CD elicits reproductive toxicity in rats, and whether such could be ameliorated by melatonin and/or Vitamin C. The CD did not affect the semen parameters while the sperm count, but not other semen parameters, was increased by melatonin in CD-treated rats. Moreover, combination of melatonin and Vitamin C in CD-treated rats increased the semen parameters. Although most of the semen parameters did not show significant change in CD-treated animals, the degeneration of the seminiferous tubules as evident from the testicular histology (not reported) suggests gonadotoxicity in these animals. It also suggests that the gonadotoxic effect of CD in male rats is independent on the hormones but might be attributed to either the direct or indirect effect of cyanide and/or its metabolite (thiocyanate) on the seminiferous tubules through induction of oxidative stress.

In the current study, residual cyanide was expected to be the only toxic substance CD fed to the rats and this might have had some adverse effects on the testis according to the histological result. However, the CD did not significantly affect the hormones of the pituitary-testicular axis in this present study. This is an indication that, despite the extent of testicular damage, the CD did not significantly disrupt the hormonal profile of pituitary-testicular axis. The mechanism of the testicular damage without a significant effect on the sperm parameters and the hormonal profile is not well understood.

  Conclusion Top

This study suggests that CD increases weight gain, thyroid hormone, and oxidative stress but has no effect on semen parameters (except sperm count) and reproductive hormones. It also suggests that melatonin and Vitamin C attenuate the effects of CD on body weight, T3, and oxidative stress. The significance of this study lies in the observation that melatonin and Vitamin C supplements (especially the former) ameliorate CD-induced increase in thiocyanate and T3 and decrease in TAC. Although the present study used an animal model, it is relevant to human because CD is very popular among people in the developing countries, especially in African and Latin American countries. Moreover, melatonin and Vitamin C are dietary supplements that are widely consumed by humans.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3]


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