Oral Administration Of Aspartame Research Articles

Aspartame, Chronic Kidney Insufficiency and Ocimum gratissimum Extract

Objective: The paired human kidneys are retroperitoneal organs responsible for the maintenance of the internal meliu of the human body. Chronic kidney disease is major cause of morbidity and mortality globally. Nutrition and life style are two main factors in the aetiopathogenesis of chronic kidney disease. Aspartame is a non-nutritive sweetener commonly consumed worldwide. Ocimum gratissimum is a naturally growing plant with some medicinal benefits. This study investigated if oral administration of aspartame could induce renal insufficiency in rat and the plausible ameliorative role of O. gratissimum extract. Methodology: Fifty animals allotted to four groups were used for the study. The control group (CN) had normal feed. The Aspartame group (Asp) had 250 mg/kg/day of aspartame for 28 days. The Aspartame low dose O. gratissimum extract group (ALO) had 250 mg/kg/day of aspartame for 28 days followed by once daily dose of the extract at 100 mg/kg for 28 days. For the Aspartame high dose O. gratissimum extract group (AHO); aspartame was administered at 250 mg/kg once daily for 28 days followed by another 28-day daily administration of the plant extract at 200mg/kg. Both aspartame and plant extract were administered orally. Periodically, blood samples were collected for biochemical analyses and kidneys harvested for histopathological examination. Results: The biochemical parameters of renal insufficiency were significantly pronounced in the Asp group but not in the AHO. Oxidative stress was pronounced in the Asp and ALO but not in group AHO at day 57. Histopathological examination of the kidney obtained from the Aspartame group revealed destruction of glomeruli. Chronic administration of aspartame in rat caused sustained renal insufficiency which was ameliorated by prolonged administration of high dose of Ocimum gratissimum extract. Thus O. gratissimum extract is beneficial to aspartame induced renal toxicity in a dose dependent manner.

Investigation of Aspartame effects on some blood parameters after oral administration in Balb-c mice

No-caloric sweeteners, such as aspartame, are widely used in various food and beverages to prevent the increasing rates of obesity and diabetes mellitus, acting as tools in helping control caloric intake. Aspartame is a methyl ester of a dipeptide used as a synthetic nonnutritive sweetener in over 90 countries worldwide in over 6000 products. It was first approved by the US Food and Drug Administration (FDA) in 1981. Aspartame is metabolized to phenylalanine, aspartic acid, and methanol. these metabolites have some health risks specially on PKU (Phenyl Ketone Urea) patients who can’t metabolize the amino acid phenyl alanine. This study aims to investigate the health effects of aspartame on Balb-c mice. 16 Balb-c mice were given physiological solution by oral gavage(control) and the study groups were given the recommended ADI (Acceptable Daily Intake) for mice (ADI = 250mg/kg/body weight) of Aspartame diluted in water for 15days, 30days. Glucose blood level, lipid profile, marker enzymes (ALT.AST.ALP, γGT) and uric acid were determined at the end of the experiment. The results of this study show that oral administration of aspartame (250mg/kg body weight) was correlated to a significant increase in the lipid profile, fasting blood glucose and some marker enzymes and this increase is time related.

Development and In‑Vitro Evaluation of Colon Specific Indomethacin Matrix Tablet for the Treatment of Inflammatory Bowel Disease

IBD is a chronic inflammatory disease of the gastrointestinal tract, particularly small and large intestine consisting of ulcerative colitis (UC) and Crohn’s disease (CD). Oral route of drug delivery is considered as the most appropriate route of drug delivery of drugs. But this route has certain limitations of poor bioavailability due to gastric degradation and increase in dose size and frequency. Therefore, the present aim of the study is to prepare and evaluate Indomethacin loaded matrix tablet using guar gum, HPMC (release controlling polymer) and citric acid to facilitate the drug solubility in the colon. A total 8 different formulations were prepared by wet granulation method and coated with Eudragit S 100 polymer a pH dependent enteric coating polymer which dissolve at colonic pH (7.4) and specifically release the drug in the colon region in sustain release fashion. The granules evaluated for its micrometrics properties and tablets were evaluated for its hardness, thickness, friability, weight variation, drug content, and in‑vitro drug release studies. The % cumulative drug release profile of all tablets was little and insignificant at pH 1.2 and 6.8. In colonic pH the coating dissolves tablets starts to release drugs. Among all the formulation the formulation F3 having guar gum and HPMC ratio 3:1 shows a maximum release of drug of 80.41±6.5%. At 24 h. The study demonstrated that the prepared tablets can release the Indomethacin in sustained release manner and helps in management of IBD with reduced side effect of the therapy. The results of this study show that oral administration of aspartame (250mg/kg body weight) was correlated to a significant increase in the lipid profile, fasting blood glucose and some marker enzymes and this increase is time related.

Effects of aspartame on the evaluation of electrophysiological responses in Wistar albino rats

Aspartame is a non-nutritive sweetener that is used predominantly in various ‘diet’ and ‘low-calorie’ products, such as beverages, instant breakfasts, desserts, breath mints, sugar-free chewing gum, vitamins, and pharmaceuticals, consumed by millions of people who are attempting weight loss, young adults and diabetic persons. On a weight basis, the metabolism of aspartame generates approximately 50% phenylalanine, 40% aspartic acid and 10% methanol. The detailed mechanisms of the effects of aspartame on the electrophysiological response are still unclear; therefore, this study was designed to clarify whether longer-term aspartame consumption has any effect on the electrophysiological response in Wistar albino rats. The oral administration of aspartame in a safe dose of 40mg/kg bodyweight/day (as recommended by EFSA, 2012) was tested in Wistar albino rats for a longer period (90 days). Electrophysiological responses, including heart rate variability (HRV) and electroencephalogram (EEG) pattern, were assessed in a folate-deficient animal model along with control animals using BIOPAC and EEG equipment (model RMS EEG–24 brain new-plus: RMS – Recorder and Medicare systems). In this study, the folate-deficient animal model was used to mimic human methanol metabolism in rats. After 90 days of aspartame treatment, a significant alteration was observable in the time domain [Mean RR (ms) SDNN (ms) RMSSD (ms) PNN50 (%)] and the frequency domain [LF, HF, and LF/HF ratio] with significantly impaired frequency and amplitude of the fronto-parietal and occipital EEG waves at p≤0.05. The results of this study clearly indicate that the oral consumption of aspartame reduced HRV, with sympathetic dominance and loss of vagal tone, and altered sympathovagal activity along with impairment of learning and memory, showing an additional effect on health within this study duration. The aspartame metabolites methanol and formaldehyde may be the causative factors behind the change observed.

Aspartame Induce Modification in Membrane Bound and Antioxidant Enzymes in Liver and Kidney of Wistar Albino Rats

Aspartame is rapidly and completely metabolized in humans and experimental animals to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite is primarily metabolized by oxidation to formaldehyde and then to formate these processes are accompanied by the formation of superoxide anion and hydrogen peroxide. This study focuses on whether the oral administration of aspartame (40 mg/kg.bw/day) for 15-days and 30- days, has any effect on membrane bound ATPase, ‘Marker’ enzymes (ALP, ALT, ACP, AST &LDH) some selective liver and kidney function parameter and antioxidant status in liver and kidney of rats. To mimic human methanol metabolism, folate deficient rats were used. After 15-days of aspartame administration, there was no change observed in kidney, however liver showed a significant decrease in membrane bound ATPase enzyme (Na + /k + , Mg 2+ & Ca 2+ ), marker enzymes (ALP, ALT, AST & LDH) and significant increase in marker enzymes (ACP), antioxidant enzyme level. However, after aspartame administration for30-days, liver and kidney both showed a significant decrease in membrane bound ATPase enzyme( Na + k + , Mg 2+ & Ca 2+ ), marker enzymes (ALP, ALT, AST & LDH) and decrease of antioxidant enzyme level in liver and significant increase in marker enzymes (ACP) and antioxidant enzyme level in kidney. This study concludes that oral administration of aspartame (40mg/kg.bw/day) for longer duration may induce oxidative stress by aspartame metabolite methanol and can damage both liver and kidney. The liver was found to be more sensitive than kidney to oxidative stress, as also reflected by the histology of liver and kidney.

Effect of long intake of aspartame on ionic imbalance in immune organs of immunized wistar albino rats

Aspartame was approved by the U.S. Food and Drug Administration (FDA) in 1981 for dry food in 1983 for soft drinks and in 1996 for all foods. In 2006, it was approved for use throughout the European Union [EFSA] on safety dose (40mg/kg b.w). The artificial dipeptide sweetener aspartame [APM; L- aspartyl-L- phenylalanine methyl ester] is present in many products especially unsweetened and sugar products. These products are frequently utilized by people trying to lose weight or patients with diabetes. Concern relating to the possible adverse effect has been raised due to aspartame metabolic components. Aspartame is rapidly and completely metabolized in humans and experimental animals to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite is primarily metabolized by oxidation to formaldehyde and then to formate these processes are accompanied by the formation of superoxide anion and hydrogen peroxide. This study focus is to understand whether the oral administration of aspartame (40mg/kg b.w.) for 90days, have any effect on membrane bound ATPase's, which may cause ionic disproportion and imbalance the homeostasis of immune organs in wistar albino rats. To mimic human methanol metabolism, folate deficient rats were used. After 90days of aspartame administration, showed a significant alteration in membrane bound ATPase's and serum ions. Excess free radical generation is confirmed by increase in lipid peroxidation and nitric oxide level. This study concludes that oral administration of aspartame (40mg/kg b.w.) for longer duration altered the homeostasis of immune organs, may cause oxidative stress in immune organs of wistar albino rats.

English

Aspartame (L- aspartyl- L-phenylalanine methyl ester) is one of the most widely artificial sweeteners consumed in so many products worldwide in various countries which added to a large variety of food, most commonly found in low calorie beverages. On metabolism in humans and experimental animals, aspartame is rapidly and completely metabolized to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite is primarily metabolized by oxidation to formaldehyde and then to formate; these processes are accompanied by the formation of superoxide anion and hydrogen peroxide. This study focus is to understand whether the oral administration of aspartame (40 mg/kg b.w) for 15, 30 and 90 days have any effect on the antioxidant status (enzymatic and non-enzymatic) in immune organs such as the spleen, thymus, lymph nodes and bone marrow of rats. To mimic human methanol metabolism, folate deficient rats were used. After 15 days of aspartame administration, animals showed a significant increase in free radical production as indicated by the increase in both enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic (reduced glutathione and vitamin C) antioxidant level along with the marked increase in lipid peroxidation and nitric oxide level. However, after repeated long term administration (30 and 90 days), the generation of reactive free radicals overwhelmed the antioxidant defense as indicated by an increase in lipid peroxidation with the decrease in antioxidants level. This study concludes that administration of aspartame even at the Food and Drug Administration permitted level its repeated exposure causes oxidative stress by altering the oxidant/antioxidant balance in immune organs of the rats and its effects also reflected in the histology of the spleen and lymph nodes Key words: Aspartame, folate- deficient, immune organs, oxidative stress.

Aspartame induces alteration in electrolytes homeostasis of immune organs in wistar albino rats

Aspartame is rapidly and completely metabolized in humans and experimental animals to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite is primarily metabolized by oxidation to formaldehyde and then to formate. These processes are accompanied by the formation of superoxide anion and hydrogen peroxide. This study is focused to understand whether the oral administration of Aspartame (40mg/kg bw) for 15 days, 30 days, and 90 days have any effect on immune organs. Damage to plasma membrane was assessed by levels of membrane-bound ATPases. Oxidative stress status was assessed by alterations in level of lipid peroxides, protein carbonyls, protein thiol and lipid-soluble antioxidant vitamin E. To mimic human methanol metabolism, folate-deficient animals were used. There was decrease in all membrane-bound ATPases activities in immune organs. Aspartame administration to rats inducing excess free radical generation is confirmed by increase in lipid peroxidation, obvious which is also again substantiated by the elevated protein carbonyl and decrease in protein thiol in this study. These excess free radical generations also decrease the cellularity (reduction in organ weight and cell count) of immune organs.

Serum biochemical responses under oxidative stress of aspartame in wistar albino rats

ObjectiveTo study whether the oral administration of aspartame (40 mg/kg body weight) for 15 d, 30 d and 90 d have any effect on marker enzymes, some selective liver and kidney function parameter, lipid peroxidation and antioxidant status in serum. To mimic human methanol metabolism, folate deficient animals were used. MethodAnimal weight, complete hemogram, marker enzyme in serum, some selected serum profile reflect liver and kidney function, plasma corticosterone level, and in serum, lipid peroxidation, nitric oxide, enzymatic and non-enzymatic antioxidant level was measured . ResultAfter 15 d of aspartame administration animals showed a significant change in marker enzymes, and antioxidant level. However, after repeated long term administration (30 d and 90 d) showed a significant change in some selected serum profile reflects liver and kidney function, along with marker enzymes, and antioxidant level. ConclusionsThis study concludes that oral administration of aspartame (40 mg/kg body weight) causes oxidative stress in Wistar albino rats by altering their oxidant/antioxidant balance.

Effect of aspartame in spinal cord and its motor behavior in Wistar albino rats.

More than 90 countries have given the artificial sweetener aspartame the green light to be used in thousands of food and beverage products. Two hundred times sweeter than sugar, aspartame allows food manufacturers to produce sweet foods they can market as “low calorie,” “diet,” or sugar-free,” appealing to hundreds of millions of consumers looking to cut sugar from their diets. Concern relating to the possible adverse effect has been raised due to aspartames metabolic components. Aspartame is rapidly and completely metabolized in humans and experimental animals to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite is primarily metabolized by oxidation to formaldehyde and then to formate these processes are accompanied by the formation of superoxide anion and hydrogen peroxide. This study focus is to understand whether the oral administration of aspartame (40 mg/kg b.w) for 90 days, have any effect on membrane bound ATPase’s, and antioxidant status in spinal cord of rats. To mimic human methanol metabolism, folate deficient rats were used. After 90 days of aspartame administration, shows a significant change in membrane bound ATPase’s and antioxidant level. This study concludes that oral administration of aspartame (40mg/kg.bw) for longer duration may cause oxidative stress in spinal cord, which didn’t have any consequence on motor behavior, but may cause other neuronal complication because oxidative stress in spinal cord can’t be ignored.

Effect of aspartame on lipid peroxidation and membrane bound enzymes in immune organs of rats

Objective: The artificial dipeptide sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester) is present in many products especially unsweetened and sugar products. These products are frequently utilized by people trying to lose weight or patients with diabetes. Concern relating to the possible adverse effect has been raised due to aspartame’s metabolic components. Aspartame is rapidly and completely metabolized in humans and experimental animals to aspartic acid (40%), phenylalanine (50%) and methanol (10%). Methanol, a toxic metabolite, is primarily, metabolized by oxidation to formaldehyde and then to formate: these processes are accompanied by the formation of superoxide anion and hydrogen peroxide. Methods: This study focus is to understand whether the oral administration of aspartame (40 mg/kg body weight) for 90 days has any effect on lipid peroxidation, nitric oxide level, membrane bound ATPases of immune organs and differential leucocyte count of rats. Results: After 90 days of aspartame administration, there was a neutrophil and lymphocyte imbalance in normal white blood cell homeostasis, a significant increase in lipid peroxidation with nitric oxide level, and an alteration of membrane bound ATPase activities, which finally decreased the cellularity (reduction in organ weight and cell count) of immune organs. Conclusion: Aspartame metabolite methanol or formaldehyde may be the causative factors behind the changes observed. This study concludes that oral administration of aspartame for longer duration may cause oxidative stress on immune organs.

Biochemical responses and mitochondrial mediated activation of apoptosis on long-term effect of aspartame in rat brain

Aspartame, an artificial sweetener, is very widely used in many foods and beverages. But there are controversies about its metabolite which is marked for its toxicity. Hence it is believed to be unsafe for human use. Previous studies have reported on methanol exposure with involvements of free radicals on excitotoxicity of neuronal apoptosis. Hence, this present study is proposed to investigate whether or not chronic aspartame (FDA approved Daily Acceptable Intake (ADI),40 mg/kg bwt) administration could release methanol, and whether or not it can induce changes in brain oxidative stress status and gene and protein expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax and caspase-3 in the rat brain region. To mimic the human methanol metabolism, Methotrexate (MTX)-treated Wistar strain male albino rats were used and after the oral administration of aspartame, the effects were studied along with controls and MTX-treated controls. Aspartame exposure resulted with a significant increase in the enzymatic activity in protein carbonyl, lipid peroxidation levels, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase and catalase activity in (aspartame MTX)-treated animals and with a significant decrease in reduced glutathione, glutathione reductase and protein thiol, pointing out the generation of free radicals. The gene and protein expression of pro apoptotic marker Bax showed a marked increase whereas the anti-apoptotic marker Bcl-2 decreased markedly indicating the aspartame is harmful at cellular level. It is clear that long term aspartame exposure could alter the brain antioxidant status, and can induce apoptotic changes in brain.

Long term of Aspartame exposure alters innate and acquired immunity of Wistar albino rats

Aim and Background: Aspartame is a non-nutritive sweetener, used in ‘diet’ and ‘low calorie’ products. Since it contains no calories, aspartame is considered a boon to health-conscious individuals. Upon ingestion, aspartame is immediately absorbed from the intestinal lumen and metabolized to phenylalanine (50%), aspartate (40%) and methanol (10%). The safety issues have been raised due to the possibility of toxicity from methanol and/or its systemic metabolite formaldehyde. Methods: This study is focus to understand whether the oral administration of aspartame (40 mg/kg.bw/day) for 90-days have any effect on immune response (innate and acquired), membrane bound ATPase of red blood cell (rbc) and antioxidant status of red and white blood cell (rbc, neutrophil and lymphocyte). Results: The present studied showed a significant alteration in immune responses, decrease in membrane bound ATPase enzymes in red blood cell. The significant increase of lipid peroxidation and nitric oxide level along with subsequent decrease in enzymatic and non- enzymatic antioxidant enzymes in red and white blood cell indicates generation of free radicals by aspartame metabolites. Conclusion: Aspartame may possibly act as chemical stressor, altered both innate and acquired immunity and generate oxidative stress in blood cells.

Effect of long-term aspartame (artificial sweetener) on anxiety, locomotor activity and emotionality behavior in Wistar Albino rats

The focus of the study is to investigate whether the long-term (90days) oral administration of aspartame (75mg/kg) has an effect in the anxiety, locomotor activity and emotionality behavior of Wistar strain male Albino rats. Aspartame releases methanol as one of its metabolites during metabolism. To mimic the human methanol metabolism, the methotrexate (MTX)-treated folate-deficient rats were used. Aspartame was administered orally to the MTX-treated animals and was studied along with controls and MTX-treated controls. The locomotor activity and emotionality behavior in the MTX-treated animals did not differ from the controls. However, the aspartame-treated MTX animals showed a marked increase in the immobilization, fecal bolus with a marked decrease in ambulation (peripheral square and central square), rearing, grooming from the control as well the MTX-treated animals. The anxiety state behavior in the MTX-treated animals did not differ from the controls. However, the aspartame-treated MTX animals showed a marked decrease in the number of open arm entry, percentage of open arm entry, time spent in open arm, percentage of time spent in open arm and number of head dips from the control as well the MTX-treated animals. It is relevant to point out that methanol released by aspartame may add an effect in the brain and that could be the additional contributing factor for the observed changes in the locomotor and their anxiety levels. This study intends to substantiate that long-term aspartame consumption can alter the behavior.

Experimental evaluation of antipyretic and analgesic activity of aspartame.

Sir, It has been reported that aspartame is biologically active and appears to relieve pain and induce mild antithrombotic effects in humans, and decrease fever in animals.[1] There is, however, a paucity of data about the biological effects of aspartame, which prompted us to undertake the present study, with a view to investigate the possible antipyretic and analgesic effects of aspartame and its comparison with conventional drugs. Albino Wistar rats of either sex (age 3-4 months, weight 200 g) were used to study the antipyretic effect, while adult mice of either sex, weighing 20-30 g, were used to study the analgesic effect. The care and maintenance of animals were as per the approved guidelines and the study was approved by the Institutional Animal Ethics Committee. Aspartame was obtained from Zydus Cadila (Ahmedabad, India). The antipyretic effect was evaluated by a 2-hour test using TAB vaccine (0.3 mL, i.p.). Rectal temperature was recorded using a clinical thermometer for a period of 2 minutes, both in basal state and at 30 minutes interval for 2 hours after the administration of vaccine. The animals were divided into five groups of 10 animals each. Group I received 0.3 mL distilled water (control). Groups II, III and IV received aspartame in a dose of 2, 4 and 8 mg/kg, respectively. Group V was administrated paracetamol (220 mg/kg). The reduction in mean rise of temperature was noted. The analgesic effect was studied by acetic acid induced writhing method.[2] Mice were divided into five groups of 10 animals each. Group I received 0.3 mL distilled water (control). Groups II, III and IV received aspartame in doses of 2, 4 and 8 mg/kg, respectively. Group V was given diclofenac 1 mg/kg by intraperitoneal route, 15 minutes prior to acetic acid injection. After intraperitoneal injection of 3% aqueous acetic acid (300 mg/kg), the mice were observed under a bell jar for a period of 20 minutes and the number of writhing episodes was noted. An average value for each group was calculated and subsequently used for comparison. In addition, protection provided by the test drugs was also calculated using the formula % protection =100 –number of writhes in experimental group × 100 number of writhes in control group Table 1 shows the effect of aspartame on rectal temperature of rats following administration of TAB vaccine. Aspartame showed a statistically significant (P < 0.001) antipyretic effect at all the doses used and this was comparable with paracetamol. Table 2 shows the effect of aspartame and diclofenac on acetic acid induced writhing episodes in mice. Aspartame in doses of 4 and 8 mg/kg showed statistically significant (P < 0.01) protection against writhing. Diclofenac, a potent analgesic compound, produced highly significant (P < 0.001) effect on writhing in doses of 1 mg/kg, i.p. Table 1 Mean rise of rectal temperature (°F) in rats Table 2 Effect of aspartame and diclofenac on acetic acid induced writhing episodes in mice In our study, we found that aspartame has highly significant antipyretic effect in doses of 2, 4 and 8 mg/kg. To our knowledge, there are no published reports of experimental evaluation of antipyretic effect of aspartame in this model. We also found that in doses of 4 and 8 mg/kg, aspartame showed a significant analgesic activity. Another study has shown that oral administration of aspartame (2-16 mg/kg) significantly increased the pain threshold against acetic acid induced writhing in mice.[3] In yet another study, it has been shown that aspartame attenuates mechanical allodynia in high doses (50 mg/kg).[4] Since this study was conducted in carrageenan-induced monoarthritis model, it appears that differences in the experimental models of evaluation of analgesic activity may account for the variation in effective doses. Interference with rheumatoid factor activity has been proposed to alleviate the pain and immobility resulting from chronic inflammation of joint.[5] In the acetic acid induced writhing method, nociceptive response involves release of endogenous substrates like bradykinin and prostanoids.[6] It is speculated that the antagonism of these chemicals may underlie the pain-relieving mechanism of aspartame. However, the exact mechanism of analgesic action of aspartame can be investigated by further pharmacological studies to overcome the limitations of current work. To conclude, aspartame is an intense nutritive sweetener, which is widely used by diabetic patients and overweight persons. Our study has demonstrated an antipyretic effect of aspartame and confirmed its analgesic effect in an experimental model. Further studies on biological effects of aspartame are required to explore its therapeutic potential as an antipyretic and analgesic agent.

Circadian Variations of Biochemical Variables in Aspartame Treated Rats

In a study of the impact of aspartame on biochemical oscillations, we examined the rhythms of blood glucose, plasma cholesterol, and protein and serum aspartate transaminase (AST) in experimental rats. Our results showed acrophase delays in glucose, total protein and advances in AST rhythms and increased mesor (in AST), amplitude (in cholesterol) and decreased amplitude values (in glucose, AST) in aspartame treated animals. Oral administration of aspartame might lead to increased levels of aspartate in brain which could alter the characteristics of biochemical variables possibly by modulating the transmission in several areas/nuclei in brain including retinohypothalamic tract (RHT) and suprachiasmatic nuclei (SCN).