Dosing-time dependent oxidative effects of an immunosuppressive drug "Mycophenolate Mofetil" on rat kidneys.

This study investigates whether the toxicity in kidneys as well as oxidative stress varied according to the dosing time of an immunosuppressive agent "mycophenolate mofetil (MMF)" in Wistar Rat. 300mg/kg of MMF was injected by intraperitonal at four different circadian stages (1, 7, 13 and 19h after light onset, HALO). Rats were sacrificed after 3days, and the kidneys were removed for determination of oxidative stress and histological analysis. Biochemical variable (creatinine, urea) was performed. Statistical analysis showed that MMF administration at 7 HALO produced a renal toxicity assessed by the significant increase in both blood creatinine and urea and antioxidant activity assessed by malondialdehyde and protein carbonyl levels indicating an induction of lipid peroxidation in oxidative damage. Whereas, at this time MMF induced a decrease the enzyme activities of renal catalase and superoxide dismutase, with a renal histopathology alterations (glomerular atrophy and lesions within proximal tubules). However, when MMF was injected in the middle of the dark-activity phase it produced a very mild renal toxicity and low oxidative stress. The obtained data indicate that the maximum of renal toxicity is observed when MMF was injected in the middle of the light- rest span in rats.

Circadian variation of cytotoxicity and genotoxicity induced by an immunosuppressive agent "Mycophenolate Mofetil" in rats.

Immunosuppressive drugs such as Mycophenolate Mofetil (MMF) are used to suppress the immune system activity in transplant patients and reduce the risk of organ rejection. The present study investigates whether the potential cytotoxicity and genotoxicity varied according to MMF dosing-time in Wistar Rat. A potentially toxic MMF dose (300 mg/kg) was acutely administered by the i.p. route in rats at four different circadian stages (1, 7, 13 and 19 hours after light onset, HALO). Rats were sacrificed 3 days following injection, blood and bone marrow were removed for determination of cytotoxicity and genotoxicity analysis. The genotoxic effect of this pro-drug was investigated using the comet assay and the micronucleus test. Hematological changes were also evaluated according to circadian dosing time. MMF treatment induced a significant decrease at 7 HALO in red blood cells, in the hemoglobin rate and in white blood cells. These parameters followed a circadian rhythm in controls or in treated rats with an acrophase located at the end of the light-rest phase. A significant, thrombocytopenia was observed according to MMF circadian dosing time. Furthermore, abnormally shaped red cells, sometimes containing micronuclei, poikilocytotic in red cells and hypersegmented neutrophil nuclei were observed with MMF treatment. The micronucleus test revealed damage to chromosomes in rat bone marrow; the comet assay showed significant DNA damage. This damage varied according to circadian MMF dosing time. The injection of MMF in the middle of the dark-activity phase produced a very mild hematological toxicity and low genotoxicity. Conversely, it induced maximum hematological toxicity and genotoxicity when the administration occurred in the middle of the light-rest phase, which is physiologically analogous to the end of the activity of the diurnal phase in human patients.

History of settlement of villages from Central Tunisia by studying families sharing a common founder Glycogenosis type III mutation.

Glycogen storage disease type III (GSD III; Cori disease; Forbes disease) is an autosomal recessive inherited metabolic disorder resulting from deficient glycogen debrancher enzyme activity in liver and muscle. In this study, we focused on a single AGL gene mutation p.W1327X in 16 Tunisian patients from rural area surrounding the region of Mahdia in Central Tunisia. This constitutes the largest pool of patients with this mutation ever described. This study was performed to trace the history of the patients' ancestries in a single region. After extraction of genomic DNA, exon 31 of AGL gene was sequenced. The patients were investigated for the hypervariable segment 1 of mitochondrial DNA and 17 Y-STR markers. We found that the p.W1327X mutation was a founder mutation in Tunisia Analysis of maternal lineages shows an admixture of autochthonous North African, sub-Saharan and a predominance of Eurasian haplogroups. Heterogeneity of maternal haplogroups indicates an ancient settlement. However, paternal gene flow was highly homogeneous and originates from the Near East. We hypothesize that the p.W1327X mutation was introduced into the Tunisian population probably by a recent migration event; then the mutation was fixed in a small region due to the high rate of consanguineous marriages and genetic drift. The screening for this mutation should be performed in priority for GSD III molecular diagnosis, for patients from the region of Mahdia and those from regions sharing the same settlement history.

Differential impact of consanguineous marriages on autosomal recessive diseases in Tunisia.

OBJECTIVES: Consanguinity is common in Tunisia. However, little information exists on its impact on recessive disorders. In this study, we evaluate the impact of consanguineous marriages on the occurrence of some specific autosomal recessive disorders and consider how other factors, such as population substructure and mutation frequency, may be of equal importance in disease prevalence. METHODS: Consanguinity profiles were retrospectively studied among 425 Tunisian patients suffering from autosomal recessive xeroderma pigmentosum, dystrophic epidermolysis bullosa, nonsyndromic retinitis pigmentosa, Gaucher disease, Fanconi anemia, glycogenosis type I, and ichthyosis, and compared to those of a healthy control sample. RESULTS: Consanguinity was observed in 341 cases (64.94%). Consanguinity rates per disease were 75.63, 63.64, 60.64, 61.29, 57.89, 73.33, and 51.28%, respectively. First-cousin marriages were the most common form of consanguinity (48.94%) with the percentages of 55.46, 45.46, 47.87, 48.39, 45.61, 56.66, and 35.90%, respectively. A very high level of geographic endogamy was also observed (93.92%), with the values by disease ranging between 75.86 and 96.64%. We observed an overall excess risk associated to consanguinity of nearly sevenfold which was proportional to the number of affected siblings and the frequency of disease allele in the family. Consanguinity was significantly associated with the first five cited diseases (odds ratio = 24.41, 15.17, 7.5, 5.53, and 5.07, respectively). However, no meaningful effects were reported among the remaining diseases. CONCLUSIONS: This study reveals a variation in the excess risk linked to consanguinity according to the type of disorder, suggesting the potential of cryptic population substructure to contribute to disease incidence in populations with complex social structure like Tunisia. It also emphasizes the role of other health and demographic aspects such as mutation frequency and reproductive replacement in diseases etiology.

Gastrointestinal toxicity of mycophenolate mofetil in rats: Effect of administration time.

This study investigates whether the intestinal toxicity of the immunosuppressive agent "mycophenolate mofetil (MMF)" varied according to the circadian dosing-time in rats. MMF (300 mg/kg) was acutely administered by i.p. route in rats at four different circadian stages (1, 7, 13 and 19 hours after light onset, HALO). The results obtained showed that MMF-induced intestinal toxicity depends on circadian dosing-time in rats. A severe toxicity in the duodenum and jejunum was observed when the drug was administered at 7 HALO compared to controls and to other circadian times. This toxicity appeared in the form of villous and Liberkhun gland atrophy and nodular inflammation. At this dosing-time, MMF induced a significant increase of phosphatase alkaline activity and a significant decrease of gut mucosa weight, protein content and disaccharidases activities. Conversely, MMF dosing at 19 HALO induced lower gut toxicity, irrespective of type of toxicity explored. These data suggest the existence of a circadian rhythm of gut toxicity for this immunosuppressive agent and the best time of gastrointestinal tolerance (chronotolerance) of this agent was observed in the middle of the dark-activity span of rats.

Circadian variation of mycophenolate mofetil pharmacokinetics in rats.

The present work aims to investigate whether the pharmacokinetics of the active metabolite mycophenolic acid (MPA) varies according to the circadian dosing-time of mycophenolate mofetil (MMF). A total of 180 male Wistar rats aged 8 weeks and synchronized for 3 weeks to 12 h light and 12 h dark were used. A single dose of 200 mg/kg of MMF was administrated in rats by i.p route at either of the four different circadian stages (1, 7, 13, and 19 Hours After Light Onset, HALO) (45 rats/circadian time). At each circadian stage, blood samples were collected at 5, 10, 15, 20, 30 min, 1 h, 1 h 30 min, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h and 24 h following drug injection. Plasma MPA concentrations were analyzed for each sample using a validated high-performance liquid chromatography (RP-HPLC) method. Tmax of MPA remained similar whatever the circadian time of injection mean Tmax=30 min). However, the peak of plasma concentration Cmax varied significantly according to the circadian dosing-time. Maximum and minimum Cmax were obtained when MMF was injected at 7 HALO (69.1 µg/ml) and at 19 HALO (22.7 ± 1.74 µg/ml) respectively. AUC0-24 varied significantly according to the circadian-time of injection (166.33 ± 10.54 mg h/L at 7 HALO vs 80.27 ± 2.33 mg h/L at 19 HALO) (p<0.05). The highest and lowest mean values of plasma clearance (CL calculated as Dos/AUC) were observed at 19 HALO (2.45 ± 0.07 L/h/kg) and at 7 HALO (1.08 ± 0.06 L/h/kg) respectively (p<0.05). Cosinor showed a circadian rhythm in the pharmacokinetic parameters Cmax, AUC0-24 and plasma clearance. The mechanism of circadian rhythm in MMF tolerance might be partly explained by the circadian variation of pharmacokinetics since the time (7 HALO) of maximum hematological and digestive toxicity corresponds to that of the lowest plasma clearance on the highest Cmax and AUC0-24 of MMF.

Circadian variation of Valproic acid pharmacokinetics in mice.

Valproic acid (VPA) is currently one of the most commonly used antiepileptic drugs. This study aims to investigate whether VPA pharmacokinetics varied according to circadian dosing-time. A single dose of VPA (350 mgkg(-1)) was administered by intraperitonally (i.p.) route to a total of 132 mice synchronized for 3 weeks to 12h light (rest span) and 12 h dark (activity span). Four different circadian times (1, 7, 13 and 19 HALO) of drug injection were used (33 mice/circadian time). At each circadian time, blood samples were withdrawn at (0 h) and at 0.083, 0.166, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2 and 3h after VPA injection. Plasma VPA concentrations were determined by an EMIT method. There were no significant differences in T(max) of VPA whatever the circadian-time of injections (T(max)=0.166 h). However, there were relevant differences in C(max) between the four circadian groups (p<0.005), it varied between 386 ± 30.86 mg L(-1) in mice treated at 7 HALO and 824 ± 39.85 mg L(-1) in mice treated at 19 HALO. The AUC(0-∞) was significantly two times higher when VPA was administered at 19 HALO as compared to the injection at 7 HALO. Drug dosing at 7 HALO resulted in highest Cl(T) value: 0.405 ± 0.006 L h(-1)kg(-1), whereas Cl(T) was significantly slower when VPA was administered at 19 HALO (0.157 ± 0.009 L h(-1)kg(-1)) (p<0.0001). The AUC(0-∞) was significantly 2-fold higher when VPA was administered at 19 HALO (2216.65 ± 138.91 mg h(-1)L(-1)) as compared to the injection at 7 HALO (864.09 ± 16.82 mg h(-1)L(-1)) (p<0.0001). Cosinor analysis showed circadian rhythm in different pharmacokinetic parameters. C(max) and AUC(0-∞) have a significant circadian rhythm with an acrophase located at 20.16 HALO ± 0.16 h (the middle of the active span) (p<0.001), whereas Cl(T) and Vd showed a significant circadian rhythm with an acrophase located respectively at 7.86 HALO ± 0.57 h and 6.13 HALO ± 0.07 h (the middle of the rest span) (p<0.001). The large circadian variation of VPA pharmacokinetic processes might be involved in the mechanisms of circadian rhythm in murine toxicity since the optimal tolerance corresponded to the time which induces lowest C(max) and AUC values.

High frequency of W1327X mutation in glycogen storage disease type III patients from central Tunisia.

Glycogen storage disease type III (GSD III) is an autosomal recessive disorder caused by the deficiency of glycogen debranching enzyme (AGL). It is characterized by hepatomegaly, progressive myopathy, cardiomyopathy and fasting hypoglycemia. Several mutations in AGL gene have been described in different populations. The W1327X mutation was reported in one Tunisian patient resident in Italy. We looked in this report to determine the frequency of W1327X mutation among Tunisian patients. The W1327X mutation was screening in 26 GSD III patients originated from various geographic locations in Tunisia. The sequence analysis revealed that among nine patients carried the W1327X mutation. Eight of them were from six unrelated families and they were originated from Mahdia (centre of Tunisia) suggesting the existence of a founder effect in this region. Taking into account historical migratory waves, screening for this mutation should be performed in priority for molecular diagnosis confirmation of GSD III in North African populations.

Chronotolerance study of the antiepileptic drug valproic acid in mice.

BACKGROUND: Valproic acid (VPA) is an antiepileptic drug widely used for the treatment of absence seizures and generalized tonic-clonic seizures. The present work aims to study whether VPA-induced toxicity varies according to the dosing-time in the 24 hour-scale. METHODS: The influence of dosing-time on tolerance to VPA was investigated in 120 male Swiss mice synchronized under a light-dark cycle (12:12). The mean VPA lethal dose was first determined to be 850 ± 0.2 mg/kg, i.p.. Such a dose was administered by i.p. route to a total of 90 mice divided in six circadian stages [1, 5, 9, 13, 17 and 21 Hours After Light Onset (HALO)] (15 mice/circadian time); 30 mice were used as control (5 mice / circadian time). RESULTS: The surviving treated mice exhibited a significant circadian variation in rectal temperature and body weight loss (p < 0.001). The least rectal temperature change and body weight loss occurred when VPA was injected at 9 HALO. Drug dosing at 9 HALO resulted in -9 % weight loss whereas drug dosing at 17 HALO was -15 % (Ø = 20.3 HALO ± 1.1 h, p ≤ 0.0001). Lethal toxicity also varied according to circadian dosing-time (χ2 = 42.1, p < 0.0001). The highest (60 %) and the lowest (6.67 %) survival rates were observed at 9 HALO and 17 HALO respectively. Cosinor analyses validated a significant circadian rhythm in survival duration with an acrophase at 8.4 HALO ± 0.75 h (p < 0.001). CONCLUSIONS: With regards to these data the optimal tolerance to VPA occurred when the drug was administered in the second half of the light-rest span of mice which is physiologically analogous to the second half of the night for human patients.

Adult Gaucher disease in southern Tunisia: report of three cases.

BACKGROUND: Gaucher disease (GD) is the most frequent lysosomal storage disorder; type 1 is by far the most common form. It is characterized by variability in age of onset, clinical signs and progression. It is usually diagnosed in the first or second decade of life with the appearance of bone pains, splenomegaly and thrombocytopenia, but the disease may be diagnosed at any age between 1 and 73 years. In the present study, we report 3 cases with late onset of GD in whom the disease was a surprise finding including one patient with Parkinson disease. This late onset is described as an adult form of Gaucher disease. FINDINGS: Molecular investigation showed mutational homogeneity in Tunisian adult patients suffering from GD. Indeed, all patients carry the p.N370S mutation: two patients at a homozygous state and one patient at compound heterozygous state. CONCLUSION: The p.N370S mutation presents a large variability in the onset of the disease and its clinical manifestation supporting the view that GD should be considered as a continuum phenotype rather than a predefined classification.

Genetic homogeneity of mutational spectrum of group-A xeroderma pigmentosum in Tunisian patients.

BACKGROUND: Xeroderma Pigmentosum (XP) is a rare autosomal recessive disorder characterized by cutaneous and ocular alterations. Eight genes, Xeroderma Pigmentosum group A (XPA) to Xeroderma Pigmentosum group G (XPG) and Xeroderma Pigmentosum group V (XPV), are known to be responsible for the disease and products of these genes are involved in the repair of deoxyribonucleic acid (DNA) lesions generated by UV radiation. Several XP patients suffer from neurological defects, found in the XPA (the most common form), D and G groups. The aim of this study was to investigate the mutational spectrum of XPA in Tunisia, in order to propose a simple tool for molecular diagnosis. METHODS: This study was carried out on six unrelated families with nine Tunisian XPA patients. Clinical features were recorded. As a previous study showed the presence of the R228X mutation in Tunisia, patients were first screened for this mutation by polymerase chain reaction-restriction fragment length polymorphism and then confirmed by direct sequencing. RESULTS: The results showed that all patients carried the XPA R228X mutation. This mutation corresponds to a C to T transition, which creates a premature stop codon at position 228, thus causing a DNA repair defect. CONCLUSIONS: The XPA R228X mutation is common in Tunisian population. This mutation is associated with a relatively moderate phenotype of the XPA. As all explored patients presented the recurrent mutation XPA R228X, a potential founder effect was searched and confirmed by haplotype analysis. Taking into account similar genetic background, investigation of this mutation should allow a cost effective and rapid diagnosis of XPA in north-African populations.

[Phenotype and mutational spectrum in Tunisian pediatric gaucher disease]. / Phénotype clinique et spectre mutationnel de la maladie de Gaucher pédiatrique en Tunisie.

BACKGROUND: Gaucher disease (GD) is a sphingolipidosis with heterogeneous phenotypic expression. The vital and / or functional prognosis may be threatened by an early visceral severe involvement in type 1 or a neurological degeneration in the more rarest neuroneupathic forms. The phenotypic and genotypic data regarding Gaucher disease are poorly known in Maghrebian countries; they are even less for pediatric forms. THE AIM of the study is to highlight the specific phenotypic and genotypic changing among the widest Gaucher pediatric cohort in the Tunisian population. METHODS: a restrospective study of a sample oh children in voluved by gaucher disease. RESULTS: Twenty one cases of GD were identified, divided into 13 cases with type 1, 5 with type 3 and 3 children with acute neurological form. The first symptoms occurred before 1 year age in one third of patients with type IGD. The clinical phenotype was severe according to the high severity score index and proportion of growth retardation. Portal hypertension was found in 8 patients. Three type 3 GD patients died before occurrence of the neurological signs. The phenotype was intermediate between the classic type 2 GD and its perinatal lethal variant. Three patients were treated with enzyme replacement therapy and 4 others had allogenic bone marrow transplantation with a favorable outcome. Three mutations dominate the genotypic spectrum of GD in this cohort. Additionally to the N370 mutation, L444P and RecNciI mutations seem to occur more frequently compared to the GD forms presenting in adulthood. CONCLUSION: This data confirm the particular severity of Gaucher disease manifesting in childhood. This was enhanced through the high frequency of severe mutations. Further studies on largest cohort are needed to more clarify the phenotypic and genotypic features of Gaucher disease in Tunisia.

Neuropharmacological screening of two 1,5-benzodiazepine compounds in mice.

This work investigates whether the two 1,5-benzodiazepine compounds: 4-(2-hydroxyphenyl)-1,5-benzodiazepin-2-one (RG0501) and Benzopyrano [4,3-c] 1,5-benzodiazepine (RG0502) have any neuropharmacological activities. Diazepam and Flunitrazepam were used as drug references. The investigational 1,5-BDZ were tested in vivo for potentiating hexobarbital-induced sleep and pentylenetetrazole (PTZ)-induced seizures. Our study demonstrated that the increase of sleep duration was significantly higher with RG0501 as compared to RG0502. However, RG0502 anticonvulsant effect was more pronounced than that of RG0501 in the range dose of 6.25-37.5 mg.kg(-1). From the 50 mg.kg(-1) dose, RG0502 offered a protection against clonic-tonic seizures as well as lethality (p< or =0.05). The results showed that the required doses to obtain a pharmacological activity were more than those of the references. This difference could be related to the lack of specific substituants responsible for the pharmacological activity in the tested compounds.